Epimerase

ABSTRACT

A novel polypeptide, acylglucosamine 2-epimerase as shown in FIG. 1 and derivatives thereof, DNA coding for said enzyme, a recombinant vector containing said enzyme, a transformant integrated thereinto said vector, a method for producing said enzyme and a method for producing N-acetylmannosamine and N-acetylneuraminic acid using renin binding protein.

This is a continuation application of U.S. patent application Ser. No.08/553,703, filed Nov. 22, 1995 now U.S. Pat. No. 5,795,767.

FIELD OF THE INVENTION

The invention relates to acylglucosamine 2-epimerase and derivativesthereof, a DNA molecule encoding the enzyme, a recombinant vectorintegrated thereinto the DNA molecule, a transformant containing thevector and a method for producing the epimerase.

The invention relates to a novel polypeptide, acylglucosamine2-epimerase and derivatives thereof having renin binding activities, aDNA molecule encoding the enzyme, a recombinant vector integratingthereinto the DNA molecule, a transformant containing the vector, amethod for producing the epimerase, an antihypertensive agent containingthe enzyme or derivative thereof as an essential component, anepimerization agent and methods for producing N-acetylmannosamine andN-acetylneuraminic acid.

BACKGROUND ART

In recent years, N-acetylneuraminic acid is noted as raw materials ofdrugs. It is known that said N-acetylneuraminic acid may beenzymatically synthesized from N-acetylmannosamine and pyruvic acidusing N-acetylneuraminic acid lyase. However, because of expensivenessand difficulty of large-scale preparation of N-acetylmannosamine, amethod for preparing N-acetylneuraminic acid by reacting inexpensiveN-acetylglucosamine and pyruvic acid in the presence ofN-acetylneuraminic acid lyase is proposed (Udo Kragl et al., AngewandteChemi-International Edition in English, 30, 827-828 (1991)). This methodutilizes that acylglucosamine 2-epimerase epimerizesN-acetyl-glucosamine to N-acetylmannosamine. However, acylglucosamine2-epimerase employed in this method exists only in a trace amount inanimal tissues and techniques of large-scale production thereof has notbeen developed. Accordingly, above-mentioned method may not be employedpractically.

On the other hand, Teshima et al. (Clinical Chemistry, 34, 2291-2294(1988)) disclose that acylglucosamine 2-epimerase is useful fordetermination of N-acetylhexosamine.

As shown above, acylglucosamine 2-epimerase is a very important enzymeand establishment of an efficient method for production thereof isearnestly desired.

It is known that acylglucosamine 2-epimerase exists in animal tissues.For example, Asis Datta (Methods in Enzymology, 41, 407-412 (1975))reported that acylglucosamine 2-epimerase existed in porcine kidney. Italso widely exists in kidney, liver, mucosal cell, submandibular gland,intestinal mucosa, colon, salivary gland, etc.

Purification of acylglucosamine 2-epimerase from animal tissues is,however, very difficult, and only crude acylglucosamine 2-epimerase isobtained up to the present. For example, Ghosh et al (Methods inEnzymology, 8, 191-195 (1966)) and Asis Datta (Methods in Enzymology,41, 407-412 (1975)) tried to isolate and purify acylglucosamine2-epimerase. However, degree of purity is low according to the report ofGhosh. According to Asis Datta, specific activity thereof is about aslow as 6 unit/mg protein.

These reports demonstrate that purification of enzyme from crude extractof porcine kidney cortex prepared by homogenizer followed by acombination of conventional purification means, such as protamineconcentration, bentonite treatment, DEAE-cellulose columnchromatography, adsorption on calcium phosphate gel, etc. is difficult.

The inventors further conduct gel filtration, hydroxyapatite,hydrophobic gel and like a variety of chromatographies andchromatofocusing in addition to said purification means, which do notlead to recovering said enzyme in a purified form due to dilution ofenzymatic activities and loss of enzymatic activities caused byinactivation of the enzyme. A trace amount of existence of the enzyme inkidney is one of reasons for difficulties of purification thereof.

Recently, preparation of heterologous proteins using microorganismsbecomes relatively easy with the progress of gene recombinationtechniques. However, because of necessity of isolation of protein forutilizing said means, materials to specify said enzyme, such as DNAprobes and antibodies may not be prepared with respect toacylglucosamine 2-epimerase which is obtained only in a crude form. Inthat case, a conventional alternative method comprises electrophoresisof a partially purified enzyme on polyacrylamide gel, blotting theenzyme on polyvinylidenedifluoride (PVDF) membrane to analyze amino acidsequence thereof, synthesizing DNA probes based on said amino acidsequence to detect a desired gene. However, with respect to this enzyme,the amino acid sequence may not be determined by said method, becauseN-terminal of acylglucosamine 2-epimerase is blocked by an unknownresidue.

As shown above, any method generally employed as gene recombinationtechniques may not be applicable to acylglucosamine 2-epimerase. A wayof producing this enzyme by gene recombination techniques has beenclosed.

It is an object of the invention to provide a method for producingacylglucosamine 2-epimerase in large quantities at low cost.

In addition, it is another object of the invention to provideacylglucosamine 2-epimerase.

Further, it is another object of the invention to provide DNA moleculescoding for acylglucosamine 2-epimerase.

Furthermore, it is another object of the invention to providerecombinant vectors into which a DNA molecule coding for acylglucosamine2-epimerase is integrated.

Furthermore, it is another object of the invention to providetransformants, wherein recombinant vectors into which a DNA moleculecoding for acylglucosamine 2-epimerase is integrated is introduced.

Furthermore, it is another object of the invention to provideantihypertensive agents.

Furthermore, it is another object of the invention to provideepimerizing agents converting N-acetylglucosamine toN-acetylmannosamine.

Furthermore, it is another object of the invention to provide methodsfor producing N-acetylmannosamine.

Furthermore, it is another object of the invention to provide methodsfor producing N-acetylneuraminic acid.

Furthermore, it is another object of the invention to provide novelpolypeptides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scheme showing a nucleotide sequence and an amino acidsequence of acylglucosamine 2-epimerase (SEQ ID NO:9.

FIG. 2 is a scheme showing a restriction map of plasmid pEPI1.

In FIG. 2, □ demonstrates pBluescript which is a vector DNA. ▪demonstrates a DNA inserted. AGE demonstrates a region of gene codingfor acylglucosamine 2-epimerase. Plac demonstrates lac promoter.

FIG. 3 is a chromatogram analyzed by HPLC of a reaction mixture modifiedby PMP. The reaction mixture was prepared by reacting extracts eitherfrom cells transformed by recombinant plasmids or cells withouttransformation in a reaction medium containing N-acetylmannosamine as asubstrate. In FIG. 3, GlcNAc demonstrates N-acetylglucosamine modifiedby PMP; ManNAc demonstrates N-acetylmannosamine modified by PMP.

FIG. 4 is a diagram showing immunological staining afterSDS-electrophoresis and western blotting of purified acylglucosamine2-epimerase derived from porcine kidney and cell extracts of E. coli.

In FIG. 4, lane 1 corresponds to purified acylglucosamine 2-epimerasederived from porcine kidney. Lane 2 corresponds to extract ofEscherichia coli. Lane 3 corresponds to extract of Escherichia colitransformed by pEPI1. Lane 4 corresponds to extract of Escherichia colitransformed by pEP114.

FIG. 5 shows elution pattern of the partially purified enzyme afterpassing through a hydroxyapatite column. Solid line demonstrates elutionof protein determined by ultraviolet absorption (280 nm); Broken linedemonstrates activities of acylglucosamine 2-epimerase. Collectedfractions are shown by arrows.

DISCLOSURE OF THE INVENTION

The inventors conducted extensive research in considering the problemsof said prior art, and purified acylglucosamine 2-epimerase in asufficient degree to allow production of antibody so as to conductcloning and production of acylglucosamine 2-epimerase using saidantibody according to gene engineering techniques. In addition,examination of a variety of biological activities of acylglucosamine2-epimerase thus obtained becomes clear that acylglucosamine 2-epimerasesurprisingly has renin binding activities. The invention has beenaccomplished based on the findings.

Thus, the invention provides an acylglucosamine 2-epimerase andderivatives thereof, DNA molecules encoding the enzyme, recombinantvectors integrated thereinto the DNA molecule, transformants containingthe vector and methods for producing the epimerase, antihypertensiveagents containing the enzyme or derivatives thereof as an essentialcomponent, epimerizating agents and methods for producingN-acetylmannosamine and N-acetylneuraminic acid according to items 1-22shown below.

Item 1. A substantially pure acylglucosamine 2-epimerase.

Item 2. An acylglucosamine 2-epimerase or a derivative thereof havingsaid enzymatic activities comprising an amino acid sequence of thefollowing formula (A) (SEQ ID NO: 1) or a amino acid sequence of formula(A) a part of which is replaced or deleted (provided that said aminoacid sequence, a part of which is replaced or deleted, hasacylglucosamine 2-epimerase activities.).

    ______________________________________                                        (A)                                                                           ______________________________________                                        Met Glu Lys Glu Arg Glu Thr Leu Gln Ala Trp Lys                               Glu Arg Val Gly Gln Glu Leu Asp Arg Val Met Ala                                 - Phe Trp Leu Glu His Ser His Asp Arg Glu His Gly                             - Gly Phe Phe Thr Cys Leu Gly Arg Asp Gly Arg Val                             - Tyr Asp Asp Leu Lys Tyr Val Trp Leu Gln Gly Arg                             - Gln Val Trp Met Tyr Cys Arg Leu Tyr Arg Lys Leu                             - Glu Arg Phe His Arg Pro Glu Leu Leu Asp Ala Ala                             - Lys Ala Gly Gly Glu Phe Leu Leu Arg His Ala Arg                             - Val Ala Pro Pro Glu Lys Lys Cys Ala Phe Val Leu                             - Thr Arg Asp Gly Arg Pro Val Lys Val Gln Arg Ser                             - Ile Phe Ser Glu Cys Phe Tyr Thr Met Ala Met Asn                             - Glu Leu Trp Arg Val Thr Ala Glu Ala Arg Tyr Gln                             - Ser Glu Ala Val Asp Met Met Asp Gln Ile Val His                             - Trp Val Arg Glu Asp Pro Ser Gly Leu Gly Arg Pro                             - Gln Leu Pro Gly Ala Val Ala Ser Glu Ser Met Ala                             - Val Pro Met Met Leu Leu Cys Leu Val Glu Gln Leu                             - Gly Glu Glu Asp Glu Glu Leu Ala Gly Arg Tyr Ala                             - Gln Leu Gly His Trp Cys Ala Arg Arg Ile Leu Gln                             - His Val Gln Arg Asp Gly Gln Ala Val Leu Glu Asn                             - Val Ser Glu Asp Gly Glu Glu Leu Ser Gly Cys Leu                             - Gly Arg His Gln Asn Pro Gly His Ala Leu Glu Ala                             - Gly Trp Phe Leu Leu Arg His Ser Ser Arg Ser Gly                             - Asp Ala Lys Leu Arg Ala His Val Ile Asp Thr Phe                             - Leu Leu Leu Pro Phe Arg Ser Gly Trp Asp Ala Asp                             - His Gly Gly Leu Phe Tyr Phe Gln Asp Ala Asp Gly                             - Leu Cys Pro Thr Gln Leu Glu Trp Ala Met Lys Leu                             - Trp Trp Pro His Ser Glu Ala Met Ile Ala Phe Leu                             - Met Gly Tyr Ser Glu Ser Gly Asp Pro Ala Leu Leu                             - Arg Leu Phe Tyr Gln Val Ala Glu Tyr Thr Phe Arg                             - Gln Phe Arg Asp Pro Glu Tyr Gly Glu Trp Phe Gly                             - Tyr Leu Asn Arg Glu Gly Lys Val Ala Leu Thr Ile                             - Lys Gly Gly Pro Phe Lys Gly Cys Phe His Val Pro                             - Arg Cys Leu Ala Met Cys Glu Glu Met Leu Ser Ala                             - Leu Leu Ser Arg Leu Ala                                                   ______________________________________                                    

Item 3. A DNA molecule coding for acylglucosamine 2-epimerase.

Item 4. A DNA molecule according to item 3 comprising a nucleotidesequence coding for amino acid sequence of the formula (A) (SEQ IDNO: 1) or a nucleotide sequence coding for an amino acid sequence offormula (A) a part of which is replaced or deleted (provided that saidamino acid sequence, a part of which is replaced or deleted, codes forpolypeptide having acylglucosamine 2-epimerase activities.).

Item 5. A DNA molecule according to item 4 comprising a nucleotidesequence of the following formula (X) (SEQ ID NO: 5)

    ______________________________________                                        (X)                                                                           ______________________________________                                        ATG GAG AAG GAG CGC GAA ACT CTG CAG GCC TGG AAG                               GAG CGT GTG GGC CAA GAG CTG GAC CGC GTG ATG GCT                                 - TTC TGG CTG GAG CAC TCC CAC GAT CGG GAG CAC GGG                             - GGC TTC TTC ACG TGC CTG GGC CGC GAC GGG CGG GTG                             - TAT GAC GAC CTC AAG TAC GTC TGG CTG CAG GGG AGG                             - CAG GTG TGG ATG TAC TGT CGC CTG TAC CGC AAG CTT                             - GAG CGC TTC CAC CGC CCT GAG CTT CTG GAT GCG GCT                             - AAA GCA GGG GGC GAA TTT TTG CTG CGC CAT GCC CGA                             - GTG GCA CCT CCT GAA AAG AAG TGT GCC TTT GTG CTG                             - ACG CGG GAC GGC CGG CCC GTC AAG GTG CAG CGG AGC                             - ATC TTC AGT GAG TGC TTC TAC ACC ATG GCC ATG AAC                             - GAG CTG TGG AGG GTG ACG GCG GAG GCA CGG TAC CAG                             - AGC GAA GCG GTG GAC ATG ATG GAT CAG ATC GTG CAC                             - TGG GTG CGA GAG GAC CCC TCT GGG CTG GGC CGG CCC                             - CAG CTC CCC GGG GCC GTG GCC TCG GAG TCC ATG GCA                             - GTG CCC ATG ATG CTG CTG TGC CTG GTG GAG CAG CTC                             - GGG GAG GAG GAC GAG GAG CTG GCA GGC CGC TAC GCG                             - CAG CTG GGG CAC TGG TGC GCT CGG AGG ATC CTG CAG                             - CAC GTC CAG AGG GAT GGA CAG GCT GTG CTG GAG AAT                             - GTG TCG GAA GAT GGC GAG GAA CTT TCT GGC TGC CTG                             - GGG AGA CAC CAG AAC CCA GGC CAC GCG CTG GAA GCT                             - GGC TGG TTC CTG CTC CGC CAC AGC AGC CGG AGC GGT                             - GAC GCC AAA CTT CGA GCC CAC GTC ATC GAC ACG TTC                             - CTG CTA CTG CCT TTC CGC TCC GGA TGG GAC GCT GAT                             - CAC GGA GGC CTC TTC TAC TTC CAG GAT GCC GAT GGC                             - CTC TGC CCC ACC CAG CTG GAG TGG GCC ATG AAG CTC                             - TGG TGG CCG CAC AGC GAA GCC ATG ATC GCC TTT CTC                             - ATG GGC TAC AGT GAG AGC GGG GAC CCT GCC TTA CTG                             - CGT CTC TTC TAC CAG GTG GCC GAG TAC ACG TTT CGC                             - CAG TTT CGT GAT CCC GAG TAC GGG GAA TGG TTT GGC                             - TAC CTG AAC CGA GAG GGG AAG GTT GCC CTC ACT ATC                             - AAG GGG GGT CCC TTT AAA GGC TGC TTC CAC GTG CCG                             - CGG TGC CTT GCC ATG TGC GAA GAG ATG CTG AGC GCC                             - CTG CTG AGC CGC CTC GCC TAG                                               ______________________________________                                    

Item 6. A recombinant vector into which a DNA molecule coding foracylglucosamine 2-epimerase is integrated.

Item 7. A recombinant vector according to item 6 comprising a nucleotidesequence coding for the amino acid sequence of the formula (A) (SEQ IDNO: 1) or a nucleotide sequence coding for an amino acid sequence offormula (A) a part of which is replaced or deleted (provided that saidamino acid sequence, a part of which is replaced or deleted, codes forpolypeptide having acylglucosamine 2-epimerase activities.).

Item 8. A recombinant vector according to item 7 comprising a nucleotidesequence coding for the amino acid sequence of the formula (X) (SEQ IDNO: 5).

Item 9. A transformant wherein a recombinant vector comprising the DNAmolecule of item 3 is introduced into said transformant.

Item 10. A method for producing acylglucosamine 2-epimerase comprisingintroducing a recombinant vector into which a DNA molecule coding foracylglucosamine 2-epimerase is integrated into a cell to form atransformant, culturing said transformant in medium to produce andaccumulate acylglucosamine 2-epimerase in culture, and collectingacylglucosamine 2-epimerase from the culture.

Item 11. An acylglucosamine 2-epimerase having renin binding activities.

Item 12. An epimerase comprising as an essential component a polypeptideas defined in (1) to (3) below:

(1) a polypeptide comprising as an essential sequence said amino acidsequence represented by the formula (A) (SEQ ID NO: 1);

(2) a polypeptide represented by the formula (A) in which at least oneof positions selected from the group consisting of 10, 13, 21, 23, 27,33, 45, 47, 51, 71, 72, 76-79, 93, 94, 101, 110, 120, 136, 137, 139,141, 142, 145, 149, 155, 159, 162, 163, 171, 173, 174, 176, 178, 187,195, 199-202, 205, 208, 212, 224, 232, 234, 237, 243, 249, 258-261, 263,266, 267, 269, 270, 272, 275, 282, 287-289, 300, 301, 309, 317, 318,328, 329, 334, 337, 348, 363, 364, 371, 392, 393, 395, 399, 401 and 402is replaced or deleted by another amino acid; or

(3) a polypeptide formed by adding Pro Ala Pro Ser Pro Ala Pro Thr ProAla Cys Arg Gly Ala Glu (SEQ ID NO: 6); Pro Ala Pro Leu Gly Ser Leu ProAla Val Pro Thr Arg Glu Gly Ser Lys (SEQ ID NO: 7); or Lys Gly Asn LysSer Trp Gln Asp (SEQ ID NO: 8 to N-terminal or C-terminal of polypeptideof formula (A); (provided that said polypeptide, in which a part offormula (A) is replaced or deleted, has acylglucosamine 2-epimeraseactivities.).

Item 13. A polypeptide of any of (1) to (3) below (provided thatpolypeptides represented by formulae (R-1), (R-2) and (R-3), listed asSEQ ID NO: 2,3, and 4, respectively, is excluded.);

(1) a polypeptide comprising as an essential sequence said amino acidsequence represented by the formula (A);

(2) a polypeptide represented by the formula (A) in which at least oneof positions selected from the group consisting of 10, 13, 21, 23, 27,33, 45, 47, 51, 71, 72, 76-79, 93, 94, 101, 110, 120, 136, 137, 139,141, 142, 145, 149, 155, 159, 162, 163, 171, 173, 174, 176, 178, 187,195, 199-202, 205, 208, 212, 224, 232, 234, 237, 243, 249, 258-261, 263,266, 267, 269, 270, 272, 275, 282, 287-289, 300, 301, 309, 317, 318,328, 329, 334, 337, 348, 363, 364, 371, 392, 393, 395, 399, 401 and 402is replaced or deleted by another amino acid; or

(3) a polypeptide formed by adding Pro Ala Pro Ser Pro Ala Pro Thr ProAla Cys Arg Gly Ala Glu (SEQ ID NO: 6); Pro Ala Pro Leu Gly Ser Leu ProAla Val Pro Thr Arg Glu Gly Ser Lys (SEQ ID NO: 7); or Lys Gly Asn LysSer Trp Gln Asp (SEQ ID NO: 8 to N-terminal or C-terminal of polypeptideof formula (A);

    ______________________________________                                        (R-1)                                                                         ______________________________________                                          Met Glu Lys Glu Arg Glu Thr Leu Gln Ala Trp Lys                             Glu Arg Val Gly Gln Glu Leu Asp Arg Val Val Ala                                  - Phe Trp Met Glu His Ser His Asp Gln Glu His Gly                             - Gly Phe Phe Thr Cys Leu Gly Arg Glu Gly Arg Val                             - Tyr Asp Asp Leu Lys Tyr Val Trp Leu Gln Gly Arg                             - Gln Val Trp Met Tyr Cys Arg Leu Tyr Arg Thr Phe                             - Glu Arg Phe Arg His Ala Gln Leu Leu Asp Ala Ala                             - Lys Ala Gly Gly Glu Phe Leu Leu Arg Tyr Ala Arg                             - Val Ala Pro Pro Gly Lys Lys Cys Ala Phe Val Leu                             - Thr Arg Asp Gly Arg Pro Val Lys Val Gln Arg Thr                             - Ile Phe Ser Glu Cys Phe Tyr Thr Met Ala Met Asn                             - Glu Leu Trp Arg Ala Thr Gly Glu Val Arg Tyr Gln                             - Thr Glu Ala Val Glu Met Met Asp Gln Ile Val His                             - Trp Val Gln Glu Asp Ala Ser Gly Leu Gly Arg Pro                             - Gln Leu Gln Gly Ala Pro Ala Ala Glu Pro Met Ala                             - Val Pro Met Met Leu Leu Asn Leu Val Glu Gln Leu                             - Gly Glu Ala Asp Glu Glu Leu Ala Gly Lys Tyr Ala                             - Glu Leu Gly Asp Trp Cys Ala Arg Arg Ile Leu Gln                             - His Val Gln Arg Asp Gly Gln Ala Val Leu Glu Asn                             - Val Ser Glu Gly Gly Lys Glu Leu Pro Gly Cys Leu                             - Gly Arg Gln Gln Asn Pro Gly His Thr Leu Glu Ala                             - Gly Trp Phe Leu Leu Arg His Cys Ile Arg Lys Gly                             - Asp Pro Glu Leu Arg Ala His Val Ile Asp Lys Phe                             - Leu Leu Leu Pro Phe His Ser Gly Trp Asp Pro Asp                             - His Gly Gly Leu Phe Tyr Phe Gln Asp Ala Asp Asn                             - Phe Cys Pro Thr Gln Leu Glu Trp Ala Met Lys Leu                             - Trp Trp Pro His Ser Glu Ala Met Ile Ala Phe Leu                             - Met Gly Tyr Ser Asp Ser Gly Asp Pro Val Leu Leu                             - Arg Leu Phe Tyr Gln Val Ala Glu Tyr Thr Phe Arg                             - Gln Phe Arg Asp Pro Glu Tyr Gly Glu Trp Phe Gly                             - Tyr Leu Ser Arg Glu Gly Lys Val Ala Leu Ser Ile                             - Lys Gly Gly Pro Phe Lys Gly Cys Phe His Val Pro                             - Arg Cys Leu Ala Met Cys Glu Glu Met Leu Gly Ala                             - Leu Leu Ser Arg Pro Ala Pro Ala Pro Ser Pro Ala                             - Pro Thr Pro Ala Cys Arg Gly Ala Glu                                      ______________________________________                                        (R-2)                                                                         ______________________________________                                          Met Glu Lys Glu Arg Glu Thr Leu Gln Val Trp Lys                                - Gln Arg Val Gly Gln Glu Leu Asp Ser Val Ile Ala                             - Phe Trp Met Glu His Ser His Asp Gln Glu His Gly                             - Gly Phe Phe Thr Cys Leu Gly Arg Asp Gly Gln Val                             - Tyr Asp His Leu Lys Tyr Val Trp Leu Gln Gly Arg                             - Gln Val Trp Met Tyr Cys Arg Leu Tyr Arg Thr Phe                             - Glu Arg Phe Arg Arg Val Glu Leu Leu Asp Ala Ala                             - Lys Ala Gly Gly Glu Phe Leu Leu Ser Tyr Ala Arg                             - Val Ala Pro Pro Gly Lys Lys Cys Ala Phe Val Leu                             - Thr Gln Asp Gly Arg Pro Val Lys Val Gln Arg Thr                             - Ile Phe Ser Glu Cys Phe Tyr Thr Met Ala Met Asn                             - Glu Leu Trp Lys Val Thr Gly Glu Met His Tyr Gln                             - Arg Glu Ala Val Glu Met Met Asp Gln Ile Ile His                             - Trp Val Arg Glu Asp Pro Ala Gly Leu Gly Arg Pro                             - Gln Leu Ser Gly Thr Leu Ala Thr Glu Pro Met Ala                             - Val Pro Met Met Leu Leu Asn Leu Val Glu Gln Leu                             - Gly Glu Glu Asp Glu Glu Met Thr Asp Lys Tyr Ala                             - Glu Leu Gly Asp Trp Cys Ala His Arg Ile Leu Gln                             - His Val Gln Arg Asp Gly Gln Val Val Leu Glu Asn                             - Val Ser Glu Asp Gly Lys Glu Leu Pro Gly Cys Leu                             - Gly Arg His Gln Asn Pro Gly His Thr Leu Glu Ala                             - Gly Trp Phe Leu Leu Gln Tyr Ala Leu Arg Lys Gly                             - Asp Pro Lys Leu Gln Arg His Ile Ile Asp Lys Phe                             - Leu Leu Leu Pro Phe His Ser Gly Trp Asp Pro Glu                             - His Gly Gly Leu Phe Tyr Phe Gln Asp Ala Asp Asp                             - Leu Cys Pro Thr Gln Leu Glu Trp Asn Met Lys Leu                             - Trp Trp Pro His Thr Glu Ala Met Ile Ala Phe Leu                             - Met Gly Tyr Arg Asp Ser Gly Asp Pro Ala Leu Leu                             - Asn Leu Phe Tyr Gln Val Ala Glu Tyr Thr Phe His                             - Gln Phe Arg Asp Pro Glu Tyr Gly Glu Trp Phe Gly                             - Tyr Leu Asn Gln Glu Gly Lys Val Ala Leu Thr Ile                             - Lys Gly Gly Pro Phe Lys Gly Cys Phe His Val Pro                             - Arg Cys Leu Ala Met Cys Glu Gln Ile Leu Gly Ala                             - Leu Leu Gln Arg Leu Gly Pro Ala Pro Leu Gly Ser                             - Leu Pro Ala Val Pro Thr Arg Glu Gly Ser Lys                              ______________________________________                                              (R-3)                                                                   ______________________________________                                                  Met Glu Lys Glu Arg Glu Thr Leu Gln Ala Trp Lys                        - Glu Arg Val Gly Gln Glu Leu Asp Arg Val Met Ala                             - Phe Trp Leu Glu His Ser His Asp Arg Glu His Gly                             - Gly Phe Phe Thr Cys Leu Gly Arg Asp Gly Arg Val                             - Tyr Asp Asp Leu Lys Tyr Val Trp Leu Gln Gly Arg                             - Gln Val Trp Met Tyr Cys Arg Leu Tyr Arg Lys Leu                             - Glu Arg Phe His Arg Pro Glu Leu Leu Asp Ala Ala                             - Lys Ala Gly Gly Glu Phe Leu Leu Arg His Ala Arg                             - Val Ala Pro Pro Glu Lys Lys Cys Ala Phe Val Leu                             - Thr Arg Asp Gly Arg Pro Val Lys Val Gln Arg Ser                             - Ile Phe Ser Glu Cys Phe Tyr Thr Met Ala Met Asn                             - Glu Leu Trp Arg Val Thr Ala Glu Ala Arg Tyr Gln                             - Ser Glu Ala Val Glu Met Met Asp Gln Ile Val His                             - Trp Val Arg Glu Asp Pro Ser Gly Leu Gly Arg Pro                             - Gln Leu Pro Gly Ala Val Ala Ser Glu Ser Met Ala                             - Val Pro Met Met Leu Leu Cys Leu Val Glu Gln Leu                             - Gly Glu Glu Asp Glu Glu Leu Ala Gly Arg Tyr Ala                             - Gln Leu Gly His Trp Cys Ala Arg Arg Ile Leu Gln                             - His Val Gln Arg Asp Gly Gln Ala Val Leu Glu Asn                             - Val Ser Glu Asp Gly Glu Glu Leu Ser Gly Cys Leu                             - Gly Arg His Gln Asn Pro Gly His Ala Leu Glu Ala                             - Gly Trp Phe Leu Leu Arg His Ser Ser Arg Ser Gly                             - Asp Ala Lys Leu Arg Ala His Val Ile Asp Thr Phe                             - Leu Leu Leu Pro Phe Arg Ser Gly Trp Asp Ala Asp                             - Tyr Gly Gly Leu Phe Tyr Phe Gln Asp Ala Asp Gly                             - Leu Cys Pro Thr Gln Leu Glu Trp Ala Met Lys Leu                             - Trp Trp Pro His Arg Gln Ala Met Ile Ala Phe Leu                             - Met Gly Tyr Ser Glu Ser Gly Asp Pro Ala Leu Leu                             - Arg Leu Phe Tyr Gln Val Ala Glu Tyr Thr Phe Arg                             - Gln Phe Arg Asp Pro Glu Tyr Gly Glu Trp Phe Gly                             - Tyr Leu Asn Arg Glu Gly Lys Val Ala Leu Thr Ile                             - Lys Gly Gly Pro Phe Lys Gly Cys Phe His Val Pro                             - Arg Cys Leu Ala Met Cys Glu Glu Met Leu Ser Ala                             - Leu Leu Ser Arg Leu Ala                                                  ______________________________________                                    

Item 14. A DNA molecule coding for polypeptide according to item 13.

Item 15. A DNA molecule according to item 14 comprising the nucleotidesequence of formula (X) (SEQ ID NO: 5).

Item 16. A recombinant vector into which a DNA molecule coding foracylglucosamine 2-epimerase according to item 14 or 15 is integrated.

Item 17. A transformant wherein a recombinant vector according to item16 is introduced into said transformant.

Item 18. A method for producing acylglucosamine 2-epimerase having reninbinding activities comprising introducing a recombinant vector intowhich a DNA molecule coding for acylglucosamine 2-epimerase having reninbinding activities is integrated into a cell to form a transformant,culturing said transformant in medium to produce and accumulateacylglucosamine 2-epimerase in culture, and collecting acylglucosamine2-epimerase from the culture.

Item 19. An antihypertensive agent comprising as an essential componentacylglucosamine 2-epimerase or a derivative thereof according to item 11or 12.

Item 20. An epimerizing agent converting N-acetylglucosamine toN-acetylmannosamine comprising as an essential component a polypeptidehaving renin binding activities.

Item 21. A method for producing N-acetylmannosamine comprising acting apolypeptide having renin binding activities on N-acetylglucosamine.

Item 22. A method for producing N-acetyneuraminic acid comprising actinga polypeptide having renin binding activities and N-acetyneuraminic acidlyase on N-acetylglucosamine and pyruvic acid.

It is known that said proteins (R-1), (R-2) and (R-3), listed as SEQ IDNO: 2,3, and 4, respectively, have renin binding activities (H. Inoue etal, J. Biochem., 110, p.493-500 (1991)). However, it is not known thatsaid proteins have acylglucosamine 2-epimerase activities. Thus,"protein having renin binding activities" employed in a method forproducing N-acetylmannosamine and a method for producingN-acetylneuraminic acid of the invention includes said proteins (R-1),(R-2) and (R-3).

The invention provides a method for producing N-acetylmannosaminecharacterized in that at least one of said proteins (R-1), (R-2) and(R-3) is applied to.

Further, the invention provides a method for producing N-acetyneuraminicacid characterized in that at least one of said proteins (R-1), (R-2)and (R-3) and N-N-acetyneuraminic acid lyase acts on N-acetylglucosamineand pyruvic acid. "Protein having renin binding activities", anessential component of epimerizing agent of the invention, comprisessaid proteins (R-1), (R-2) and (R-3).

Therefore, the invention provides an epimerizing agent convertingN-acetylglucosamine to N-acetylmannosamine comprising as an essentialcomponent at least one selected from the group consisting of saidproteins (R-1), (R-2) and (R-3).

The novel polypeptides of the invention are useful as acylglucosamine2-epimerase.

Since said items 1-10 are enough as long as they have acylglucosamine2-epimerase activities, irrespective of existence of renin bindingactivities, items 1-10 are hereinafter referred to as "first invention".

In addition, said items 11-22 relates to proteins having bothacylglucosamine 2-epimerase activities and renin binding activities, andhereinafter referred to as "second invention".

Further, "the invention" includes first invention and second invention.

At least some of acylglucosamine 2-epimerases of the invention haveacylglucosamine 2-epimerase activities and renin binding activities.Said epimerases include all proteins having acylglucosamine 2-epimeraseactivities, irrespective of renin binding activities thereof.

According to the invention, cells transformed by recombinant vector intowhich DNA molecule coding for acylglucosamine 2-epimerase is integratedare not specifically limited. Example of such cells are Escherichiacoli, Bacillus subtilis, Pseudomonas aeruginosa, Actinomycetes, Lacticacid bacteria and like bacteria, fungi, yeast and like eucaryoticmicroorganisms, mouse cells, rat fibroblast, plant cells and like cellsas long as cells allow stable retainment and function of plasmids.

For carring out the invention, skilled artisan may suitably select amethod for introducing a plasmid into which a DNA molecule coding foracylglucosamine 2-epimerase is integrated into said cell, according tothe type of cells. For example, introduction of plasmid into E. coli maybe carried out according to a method of Hanahan (DNA Cloning, Vol.1,p.109-136 (1985)).

The invention first isolates acylglucosamine 2-epimerase in a formsubstantially free of impurities and discloses a method for productionthereof. The invention first discloses that at least a part of saidepimerases have renin binding activities. The second invention includespolypeptides having both acylglucosamine 2-epimerase activities andrenin binding activities irrespective of amino acid sequence thereof.For example, the invention includes DNA molecules in which several toone handred and tens of nucleotides at 5' terminal and/or 3' terminalare eliminated by exonuclease, and derivatives of said enzyme encoded bysaid DNA in which several to tens of amino acids at N-terminal and/orC-terminal are eliminated, as long as said derivatives retain saidenzymatic activities. Further, the invention includes polypeptides inwhich at least one amino acid in the amino acid sequence thereof isdeleted or replaced by another amino acid according to known pointmutation methods, as long as said polypeptides retain saidacylglucosamine 2-epimerase activities.

Positions of polypeptides represented by formula (A) allowingreplacement and/or deletion are not specifically limited to, but include10, 13, 21, 23, 27, 33, 45, 47, 51, 71, 72, 76-79, 93, 94, 101, 110,120, 136, 137, 139, 141, 142, 145, 149, 155, 159, 162, 163, 171, 173,174, 176, 178, 187, 195, 199-202, 205, 208, 212, 224, 232, 234, 237,243, 249, 258-261, 263, 266, 267, 269, 270, 272, 275, 282, 287-289, 300,301, 309, 317, 318, 328, 329, 334, 337, 348, 363, 364, 371, 392, 393,395, 399, 401 and 402.

Further, the invention include polypeptides to which several to tens ofamino acids at N/terminal and/or C-terminal are added, as long as saidpolypeptides retain said acylglucosamine 2-epimerase activities.

Donors of nucleic acid molecule coding for acylglucosamine 2-epimeraseemployed in the invention are not specifically limited to, but includeanimal tissues having said enzymatic activities, such as porcine andhuman kidneys, kidney, liver, mucosal cell, submandibular gland,intestinal mucosa, colon, salivary gland, etc. of human or rat. Saidnucleic acid molecules are obtained from the animal tissues. The nucleicacid molecules include DNAs and RNAs, preferably RNAs.

RNAs coding for acylglucosamine 2-epimerase may be obtained according toa method of Chomczynski et al (Analytical Biochemistry, 162, 156-159,(1987)). RNAs are also obtained as RNAs with polyadenylate tail (poly Atail) according to a method described in Molecular Cloning SecondEdition Val.1, sections 7.26-7.29. cDNAs coding for acylglucosamine2-epimerase may be easily obtained by using the RNAs with poly A tail.

Conversion of RNA to cDNA may be carried out, for example, according toCurrent Protocols in Molecular Biology, Vol.1, 5.5.1-5.5.10 (1990). Theconversion may also be carried out by using commercially available cDNAsynthesis kit (eg. product of Amersham, Stratagene, etc).

cDNA library may be constructed by inserting cDNA into some vectors, forexample, phage vectors and plasmid vectors. Construction of cDNA librarymay be carried out according to a method described in Molecular CloningSecond Edition Vol.2, sections 8.1-8.86.

Means to select transformant retaining cDNA coding for acylglucosamine2-epimerase from cDNA library constructed are needed. With respect tosaid enzyme, purified enzyme with required level in the field ofrecombination of gene was not obtained as stated above. The inventorsobtained acylglucosamine 2-epimerase purified enough to produce antibodyaccording to the following method.

First, acylglucosamine 2-epimerase is partially purified according tothe method of Asis Datta (Methods in Enzymology, 41, 407-412 (1975)). Inthe following purification procedure, fractions with high activities andsmall amount of proteins, i.e., only fractions whose specific activityis higher than specific activity of sample before elution of column arecollected, because fractions having activities spread in the process ofpurification. Specifically, it is found that said enzyme is purified byapplying said partially purified sample to hydroxyapatite column andion-exchange chromatography in this sequence to collect the elutedfraction with highest enzymatic activities, followed by separationthereof by ion-exchange chromatography repeated two times. The enzymefraction is further purified by HPLC with reverse-phase column(μBondasphere, product of Millipore) to obtain said purified enzymeprotein substantially free of impurities. The reverse-phase HPLC ishardly employed for purification of enzymes, since enzymes are easilyinactivated during purification. Acylglucosamine 2-epimerase also losesenzymatic activities thereof by said purification, but maintainelicitation ability to produce antibodies. Therefore, antibodiesspecifically reacted with said enzyme protein are obtained by immunizingrabbit with acylglucosamine 2-epimerase purified by HPLC.

According to said purification steps, for example, 10.5 mg of saidpurified enzyme protein is obtained from 5.6 kg of porcine kidneycortex.

The DNA molecule coding for acylglucosamine 2-epimerase of the inventionmay be isolated from constructed cDNAs. The isolation may be carried outby detecting said enzyme with the antibodies using λgt11, λZAP and likevectors as expression vector. The transformant retaining DNA coding foracylglucosamine 2-epimerase produces acylglucosamine 2-epimerase, whenexposed by isopropyl-β-D-thiogalactopyranoside (IPTG). The transformantretaining DNA coding for said enzyme may be selected and obtained bybinding this to antibody, to which anti-rabbit antibody is bound,followed by reacting the complex with5-bromo-4-chloro-3-indolylphosphate solution and nitroblue tetrazoliumsolution.

When resulting DNA coding for acylglucosamine 2-epimerase is insertedinto plasmid DNA, E. coli containing said DNA is cultured in a suitablemedium to obtain acylglucosamine 2-epimerase. When μZAP is used as avector, said DNA may be excised cut as a plasmid into vector bysimultaneous infection of obtained phage containing said DNA and f1helper phage. A novel recombinant plasmid containing DNA, for example,represented by formula (X) having 1.2 kbp, coding for acylglucosamine2-epimerase may be obtained according to the series of operations.

Cells such as Escherichia coli are transformed by using plasmid insertedtherein a DNA fragment thus obtained coding for acylglucosamine2-epimerase. In order to produce acylglucosamine 2-epimerase fromtransformed cells, transformed cells are cultured in the followingconditions to obtain mass of cells.

Culture conditions are different depending on types of cellstransformed, and are easily determined in accordance with types ofcells. For example, E. coli may be cultured by conventional solidculture method, but is preferably cultured by liquid culture method. Inaddition, taking E. coli as an example, media employed for culturecontain carbon sources, nitrogen sources, inorganic compounds and othernutrient components. Any one of synthetic medium, semisynthetic medium,natural medium and like medium generally employed for culturing bacteriamay be used. Examples of carbon sources for said media are glucose,fructose, invert sugar, starch, saccharified starch, sorbitol, glycerol,and like carbohydrate solution, pyruvic acid, malic acid, succinic acid,and like organic acids. Examples of nitrogen sources are ammoniumsulfate, ammonium chloride, ammonium nitrate, ammonium phosphate,ammonium hydroxide, ammonium tartrate, ammonium acetate, urea, etc.Substances employed as both carbon and nitrogen sources include peptone,yeast extract, meat extract, corn steep liquor, etc. Examples ofinorganic compounds are potassium dihydrogenphosphate, dipotassiumhydrogenphosphate, sodium dihydrogenphosphate, disodiumhydrogenphosphate, magnesium phosphate, magnesium chloride, potassiumchloride, sodium chloride, ferrous sulfate, ferric sulfate, ferricchloride, manganese sulfate, manganese chloride, etc.

Culture time and culture temperature of transformed cells are notspecifically limited. Taking Escherichia coli as an example of saidcells, culture is conducted at generally 20-42° C., preferably 30-37°C., for generally 4-48 hours, preferably 8-14 hours. Under theseconditions, conventional shake culture or aeration agitation culture iscarried out.

When DNA coding for acylglucosamine 2-epimerase of the invention isbound to a suitable vector to transform a suitable host cell, oxygenconcentration of inside or outside of transformed host cell is increasedleading to efficient production of said enzyme.

The vector employed in the invention comprises the following elements.Specifically, the vector comprises promoter placed in a correctorientation and position to express DNA coding for acylglucosamine2-epimerase of the invention and translation activating sequence. Anyvector comprising these elements may be employed, but preferably arevectors comprising suitable selected marker and multicopy vectors, whichinclude pBluescript, pUC18, pUC19, pKK223-3 and pTrc99A, etc. When thesevectors are employed, intracellular concentration of acylglucosamine2-epimerase may be elevated by adding about 0.01 mM-100 mM, preferablyabout 0.1 mM-10 mM of isopropyl-β-D-thiogalactopyranoside to culturemedium. Further, pPL-lambda, heat-induced expression vector, may beemployed. When employing this vector, intracellular concentration ofacylglucosamine 2-epimerase may be increased by elevating temperature ofculture medium to 40-45° C.

Further, increase of productivity and efficiency of said enzyme may beaccomplished by deleting terminal DNA of said DNA coding foracylglucosamine 2-epimerase to increase intracellular concentration ofacylglucosamine 2-epimerase. For example, production efficiency ofacylglucosamine 2-epimerase may be further increased by degradation ofsaid DNA molecule at 5' and/or 3' terminal with exonuclease, etc., whilemaintaining acylglucosamine 2-epimerase activities.

In addition, productivity of said enzyme may be increased bysite-specific mutation or random mutation to replace or delete said DNAat an internal region. For example, site-specific mutation may beintroduced by integrating said DNA molecule into pBluescript, M13 andlike vectors capable of becoming single strand to prepare single strandDNA containing said DNA molecule; annealing oligonucleotides containingsequence to be mutated or deleted to said single strand DNA; andconducting primer elongation reaction utilizing said oligonucleotides asa primer in the presence of deoxyribonucleotide triphosphate, ATP,Klenow fragment and T4 ligase.

Acylglucosamine 2-epimerase may be extracted from cultured cells.Extraction may be carried out according to conventional enzymeextraction methods. For example, said enzyme may be collected fromsupernatant by crushing cells with ultrasonic treatment, a variety ofmechanical treatments, enzymatic treatments and like methods, followedby separating insoluble matter by centrifugation. The collected crudeenzyme may be purified by suitably combining conventional enzymepurification methods. For example, a great amount of purifiedacylglucosamine 2-epimerase may be obtained by nucleic acid removaltreatment, ammonium sulfate treatment, diatomaceous earth treatment,ion-exchange chromatography, etc.

The Escherichia coli strain containing pEPI1 of FIG. 2 integratedthereinto the DNA molecule of FIG. 1 is deposited in National Instituteof Bioscience and Human-Technology Agency of Industrial Science andTechnology and accession number: FERM BP-4602 is given.

The inventors conducted extensive research on biological activities ofpolypeptide of formula (A) derived from porcine kidney obtained insecond invention and make clear that said enzyme has not onlyacylglucosamine 2-epimerase activities but also renin bindingactivities. "Renin binding activities" mean that said polypeptide bindsto renin resulting in inhibition of renin activities. The polypeptide offormula (A) is obtained according to the method for producingacylglucosamine 2-epimerase of the invention. In contrast, renin bindingprotein from porcine kidney of formula (R-3) is obtained according to amethod disclosed by Inoue et al (H. Inoue et al., J. Biochem., 110, p.493-500, (1991)). The two proteins (A) and (R-3) obtaining by differentpurification method are very similar proteins except that they havedifferent amino acids at positions 149, 289, 317 and 318. As shown inthe following examples, the polypeptide (A) obtained according to theinvention and epimerase from rat kidney have renin binding activities,which indicates that, with respect to at least part of proteins,acylglucosamine 2-epimerase activities and renin binding activities areinseparable. Accordingly, known renin binding proteins (R-1), (R-2) and(R-3) will have epimerase activities. When renin binding proteins haveacylglucosamine 2-epimerase activities, said renin binding proteins maybe employed as epimerizing agents catalizing the conversion reactionfrom N-acetylglucosamine to N-acetylmannosamine.

The protein of formula (A) is different from known proteins (R-1), (R-2)and (R-3) in any one of positions 10, 13, 21, 23, 27, 33, 45, 47, 51,71, 72, 76-79, 93, 94, 101, 110, 120, 136, 137, 139, 141, 142, 145, 149,155, 159, 162, 163, 171, 173, 174, 176, 178, 187, 195, 199-202, 205,208, 212, 224, 232, 234, 237, 243, 249, 258-261, 263, 266, 267, 269,270, 272, 275, 282, 287-289, 300, 301, 309, 317, 318, 328, 329, 334,337, 348, 363, 364, 371, 392, 393, 395, 399, 401 and 402. However, theseproteins have acylglucosamine 2-epimerase activities and renin bindingactivities so that these positions are not essential for biologicalactivities and may be replaced or deleted. Further, addition of tens ofamino acids to the proteins at N-terminal, C-terminal does not affectexpression of activities of the proteins. The inventors, in fact,obtained an epimerase obtained by adding amino acids, Lys Gly Asn LysSer Trp Gln Asp, to the polypeptide of formula (A) at N-terminal. It isconfirmed that said epimerase has sufficient enzymatic activities.

The invention discloses a method for collecting acylglucosamine2-epimerase produced intracellularly in large amounts comprisingseparating a DNA molecule coding for acylglucosamine 2-epimerase fromanimal tissues, preparing a recombinant plasmid containing said DNAmolecule, introducing said plasmid into E. coli and like host cell totransform the cell and culturing the transformant.

Renin produces angiotensin I by hydrolyzing angiotensinogen. AngiotensinI is further converted to angiotensin II with angiotensin convertingenzyme to express strong hypertensive action by directly constrictingsmooth muscle of peripheral blood vessel. Angiotensin II also acts onadrenal gland zona glomerulosa to accelerate secretion of aldosterone.As shown above, renin-angiotensin system plays an important role onregulation of blood pressure. Therefore, inhibitors of renin-angiotensinsystem are developed and widely employed as anti-hypertensive agent.Since acylglucosamine 2-epimerase of the invention binds to reninleading to inhibit activities thereof, acylglucosamine 2-epimerase isuseful as an anti-hypertensive agent.

Renin binding proteins have acylglucosamine 2-epimerase activities.Accordingly, renin binding proteins may be employed as epimerizing agentconverting N-acetylglucosamine to N-acetylmannosamine. When reninbinding proteins act on N-acetylglucosamine, N-acetylmannosamine isobtained. Further, when renin binding proteins and N-acetylneuraminicacid lyase act on N-acetylglucosamine and pyruvic acid,N-acetylneuraminic acid may be obtained by convertingN-acetylglucosamine to N-acetylmannosamine with the action of reninbinding protein, followed by binding N-acetylmannosamine to pyruvicacid.

According to the invention, the outstanding effects as shown below areexerted.

(1) Highly purified acylglucosamine 2-epimerase may be obtained in largeamounts with low costs.

(2) Because microorganisms are employed as starting materials, themethod of the invention is not limited with respect to supply of rawmaterial unlike conventional method using animal tissues as rawmaterial. Therefore, production thereof may be carried out at a desiredplace in required amounts in any time.

(3) Because of high productivity of acylglucosamine 2-epimerase, saidenzyme may easily be isolated.

(4) Because high-purity acylglucosamine 2-epimerase may be obtained withlow costs, N-acetylneuraminic acid and N-acetylmannosamine may beproduced with low costs.

(5) Because high-purity acylglucosamine 2-epimerase may be obtained, itmay be employed as assay of N-acetylneuraminic acid andN-acetylhexosamine.

(6) Antihypertensive agents are obtained by using proteins having reninbinding activities of the invention.

(7) Because proteins having renin binding activities also haveacylglucosamine 2-epimerase activities, said proteins may be employed asepimerizing agents converting N-acetylglucosamine toN-acetylmannosamine.

(8) N-acetylneuraminic acid may be efficiently obtained by reactingproteins of the invention with renin binding activities andN-acetylneuraminic acid lyase with N-acetylglucosamine and pyruvic acidoptionally under alkaline conditions.

EXAMPLES

The invention will be described below in greater detail using examples,but the invention is in no way limited to the examples.

Example 1

Production of Acylglucosamine 2-epimerase from Porcine Kidney

(1) Purification of Acylglucosamine 2-epimerase from Porcine Kidney

Freshly obtained porcine kidney cortex (5.6 kg) supplemented with 12liter of 3 mM phosphate buffer (pH 7.6) was homogenized withhomogenizer. After obtaining supernatant (11 liter) by sequentialcentrifugation (10,000 rpm, 200 ml/min), cooled distilled water equalvolume to the supernatant was added thereto. The resulting mixture wassufficiently stirred and then sequentially centrifuged (10,000 rpm, 200ml/min) to obtain kidney extract as supernatant. The kidney extract wastreated by protamine concentration, bentonite treatment, DEAE-cellulosecolumn chromatography and calcium phosphate gel according to Asis Datta(Methods in Enzymology, 41, 407-412 (1975)) for purification ofacylglucosamine 2-epimerase to obtain 387 mg of partially purifiedenzyme.

Said partially purified enzyme was applied to hydroxyapatite column(inner diameter 26 mm×length 95 mm; WAKO PURE CHEMICAL CO., LTD.)equilibrated with 10 mM phosphate buffer (pH 7.6) and eluted with thesame buffer. Fractions with said enzymatic activities were eluted inwidely spread condition according to the treatments of the invention(see, FIG. 5). The 12th to 17th fractions (18 ml×6=108 ml) having saidenzymatic activities were treated with salting out techniques usingammonium sulfate to collect fractions saturated with 0-80 % by weight ofammonium sulfate. The fractions were dialyzed against 20 mM phosphatebuffer (pH 7.6) to give 75.6 mg of dialyzed enzyme. The enzyme wasfurther purified by ion-exchange chromatography in the followingconditions to purify the enzyme.

Q-Sepharose (PHARMACIA), one of ion-exchange resins, was filled in acolumn (inner diameter 26 mm×length 95 mm). The column was equilibratedby 20 mM phosphate buffer (pH 7.6; 500 ml). Said partially purifiedenzyme (75.6 mg) was adsorbed on the column and eluted by lineargradient using phosphate buffer (pH 7.6) containing 100 mM to 300 mM ofpotassium chloride. A main peak corresponding to a protein eluted with apotassium chloride concentration of about 180 mM (198 ml) was collected,and then concentrated and dialyzed. The dialyzed enzyme (23.0 mg) wasapplied to Mono Q column (PHARMACIA) to adsorb said enzyme on thecolumn. The adsorbed enzyme was eluted by linear gradient usingphosphate buffer (pH 7.6) containing 200 mM to 300 mM of potassiumchloride. A peak corresponding to a protein eluted with a potassiumchloride concentration of about 220 mM was collected, and then desaltedby gel filtration column. An activity of the enzyme (15.9 mg) thusobtained was 21 units/mg protein as a specific activity, which is 3.5time as high as the activity (purity) of the protein (6 units/mgprotein) reported by Asis Datta (Methods in Enzymology, 41, 407-412(1975)).

Further, the resulting enzyme was purified to removelow-molecular-weight materials and trace amount of impurities by HPLCusing reverse phase column. μBondasphere 5 μC4-300 Å (inner diameter 3.9mm×length 150 mm) (MILLIPORE) was employed as reverse phase column. Saidpurified enzyme (2 mg) was subjected to the column equilibrated with0.1% (V/V) TFA aqueous solution. Said purified enzyme retained in thecolumn was eluted by linear gradient using 0.1% (V/V) TFA aqueoussolution containing 0 to 80% (V/V) of acetonitrile. A main peak proteindetermined by ultraviolet absorption (280 nm) was collected and dried invacuo. Said procedure was repeated 6 times to obtain the enzyme (10.5mg). The enzyme thus obtained loses biological activities but issubstantially free of impurities.

(2) Antibodies Specifically Bound to Acylglucosamine 2-epimerase

Nine weeks old rabbits (JAPANESE WHITE) were immunized with saidpurified acylglucosamine 2-epimerase (5.2 mg). Blood of said rabbit wasgathered partially and wholly to obtain 150 ml of antiserum. IgG waspurified from the antiserum using Protein A Sepharose according to thedescription on page 24 of New Biochemical Experimental Course 12, Ed.Japan Biochemistry Organization, Molecular Immunology III, (1992).

Example 2

Cloning of cDNA Coding for Acylalucosamine 2-epimerase

(1) Production of mRNA from Porcine Kidney

Kidney cortex was cut out from porcine kidney. RNAs (4.9 mg) wereobtained from 2 g of kidney cortex according to the method ofChomczynski et al (Analytical Biochemistry, 162, 156, (1987)). RNAs withpoly(A) tail (67 μg) were then obtained by adsorbing said RNA onoligo-dT-cellulose column, followed by elution thereof.

(2) Preparation of cDNA Library

A cDNA library was prepared from the RNAs with poly(A) tail (5 μg) thusobtained using a ZAP-cDNA synthesis kit (STRATAGENE). The libraryobtained was 4.5×10¹² of plaque forming unit.

(3) Screening of Gene of Acylglucosamine 2-epimerase

Screening of recombinant containing DNA coding for acylglucosamine2-epimerase was carried out according to immunostaining method usingantibody of example 1 and picoBlue Immunoscreening Kit (STRATAGENE).

64 positive phages were obtained by screening 1,200,000 of plaquesaccording to said method. Optionally 12 strains were selected from saidpositive phages. E. coli was infected by said strains with f1 helperphage so as to integrate cDNAs into plasmids. E. coli strain withplasmid having acylglucosamine 2-epimerase activities was selected. Theplasmid was taken out from E. coli selected to prepare a recombinantplasmid pEPI1 (4.3 kbp) containing insertional fragment (1.4 kbp).Restriction map of pEPI1 is shown in FIG. 2.

The E. coli into which pEPI1 (4.3 kbp) was introduced was depositedunder accession number FERM BP-4602 in National Institute of Bioscienceand Human-Technology Agency of Industrial Science and Technology on Mar.11, 1994.

(4) Determination of Acylglucosamine 2-epimerase Activities

In order to determine acylglucosamine 2-epimerase activities in E. coli,E. coli XL1-Blue was inoculated in LB medium (1% peptone, 0.5% yeastextract, 1% NaCl, pH 7.0) containing 100 mg/liter of ampicillin and thenshake-cultured. Further, for efficient production of said enzyme,isopropyl-β-D-thiogalactopyranoside was added to the medium within afinal concentration of 1 mM when starting culture. The resulting mediumwas cultured at 37° C. for 12 hours and then centrifuged (5,000 rpm, 10minutes) to obtain pellet of cells. Cell extract was obtained byultrasonic disruption of cells. The cell extract was reacted at 37° C.for 30 minutes at a final volume of 0.5 ml in the presence of 40 mM ofN-acetylmannosamine (NACALAI TESQUE) or 40 mM of N-acetylglucosamine(NACALAI TESQUE), 4 mM of adenosine-triphosphate (KOJIN Co., Ltd.), 10mM of magnesium chloride and 100 mM of Tris-HCl buffer (pH 7.5). Theresulting reaction mixture was boiled for 3 minutes in boiled water tostop the reaction. The enzymatic activities were determined by assayingN-acetylmannosamine or N-acetylglucosamine of reaction supernatantprepared by centrifugation (12,000 rpm, 5 minutes) of said reactionmixture. In the determination, it is difficult to determineN-acetylmannosamine and N-acetylglucosamine as they are by HPLC. Inorder to convert these compounds to 1-phenyl-3-methyl-5-pyrazolone (PMP)derivatives according to the method of Honda et al (AnalyticalBiochemistry, 180, 351-357 (1989)), 10 μl of said reaction supernatantwas removed to 1.5 ml volume of microtube, to which 50 μl of 0.5M PMPmethanol solution and 50 μl of 0.3M NaOH aqueous solution was added,subsequently the mixture was reacted at 70° C. for 30 minutes. Thereaction mixture was cooled at room temperature for 10 minutes, and thenneutralized with 150 μl of 0.1M hydrochloric acid aqueous solution. Tothe neutralized solution was added 200 μl of chloroform, and the mixturewas mixed to separate unreacted PMP in chloroform layer to obtainPMP-N-acetylhexosamine in aqueous layer. After removing chloroformlayer, the aqueous layer was dried in vacuo. The dried residue wasdissolved in 250 μl of distilled water, 10 μl of which was injected toHPLC for separation and determination. HPLC analysis was carried outusing LC-6A system (SHIMADZU CORP.) and column (Cosmosil 5C18-AR (innerdiameter 6.0 mm×length 150 mm), NACALAI TESQUE) with a mixture ofacetonitrile and 50 mM phosphate buffer (pH 7.0) (2:8) as mobile phaseat a rate of 1 ml/min. Detection were performed by absorption amount ofultraviolet at 245 nm. The enzymatic activities are shown as unit. Oneunit is defined as activity to produce 1 μmol of N-acetylglucosamine per1 minute from the reaction of N-acetylmannosamine as substrate.

Consequently, when N-acetylmannosamine was contained in the reactionmixture, N-acetylmannosamine was converted to N-acetylglucosamine in thepresence of cell extract (see FIG. 3). Similarly, N-acetylglucosaminewas converted to N-acetylmannosamine in the presence of cell extract. Ineach reaction, N-acetylglucosamine: N-acetylmannosamine reached aequilibrium condition of 75: 25. No conversion was observed withoutaddition of said cell extract to said mixture. Furthermore, antibodiesdirected to purified acylglucosamine 2-epimerase was reacted with a bandof 45,000 daltons in western blotting. The test results, as a whole,demonstrate that cloned gene produce protein corresponding toacylglucosamine 2-epimerase. Further, acylglucosamine 2-epimeraseactivities of the cell extract correspond to 10 unit production per 1liter of culture medium, showing that specific activities of cellextract was 0.03 U/mg, which is similar to extract of porcine kidneycortex. The results demonstrate that acylglucosamine 2-epimerase,conventionally obtained only from animal tissues, may be produced bymicroorganisms transformed by plasmid pEPI1.

(5) Determination of Nucleotide Sequence of Acylglucosamine 2-epimerase

A necleotide sequence of 1.4 kbp of DNA fragment was determined bydideoxy method whose basic principle was a process described by Sangeret al (Proceedings of The National Academy of Sciences of the UnitedStates of America, 74, 5463-5467 (1977)). Because a vector employed inpEPI1 was pBluescript capable of becoming single strand, deletion mutantof the vector was prepared, and then nucleotide sequence thereof wasdetermined. The nucleotide sequence coding for acylglucosamine2-epimerase-is shown in FIG. 1 (SEQ ID NO:9). Further, amino acidsequence of polypeptide obtained by translation of said nucleotidesequence coding for the enzyme is shown simultaneously.

Example 3

Production of Acylglucosamine 2-epimerase by Microorganisms

(1) Construction of Plasmid Producing Acylglucosamine

2-epimerase with High Efficiency

Highly efficient production of acylglucosamine 2-epimerase bymicroorganisms become possible by construction of deletion plasmid ofpEPI1. The plasmid pEPI1 (20 μg) in 500 μl solution was cut at 37° C.for 4 hours with restriction enzymes of 100 units of SacI and 100 unitsof XbaI. The mixture treated by restriction enzymes was treated at 75°C. for 15 minutes, extracted by phenol/chloroform (1:1) and thenprecipitated with ethanol. The precipitate was dissolved in sterilizedwater at a concentration of 1 μg/μl. Tens of deletion plasmids wereprepared from the solution using ExoIII/Mung Deletion Kit (STRATAGENE),in which pEP114 produced acylglucosamine 2-epimerase with highefficiency. E. coli XL1-Blue transformed by plasmid pEP114 was culturedin LB medium containing 100 μl/ml of ampicillin and 1 mM ofisopropyl-β-D-thiogalactopyranoside at 37° C. for 12 hours. Cell extractwas prepared from cells of said culture. Acylglucosamine 2-epimeraseactivities of cell extract correspond to about 1,000 units per 1 literof culture medium, and specific activities thereof was 1.6 U/mg, whichwas 53 times as much as extract of porcine kidney cortex.

(2) Production of Acylglucosamine 2-epimerase

The plasmid pEP114 provides useful means for producing a larger amountof acylglucosamine 2-epimerase in E. coli. Culture of E. coli to obtainacylglucosamine 2-epimerase is much easier than preparation thereof fromanimal tissue.

Culture thereof was carried out by inoculating E. coli XL1-Bluetransformed by plasmid pEP114 in two shaking flasks (2-liter volume)containing 500 ml of LB medium (1% peptone, 0.5% yeast extract, 1% NaCl,pH 7.0) containing 100 mg/liter of ampicillin and 1 mM IPTG, thenshake-cultured the flasks. After culture at 37° C. for 12 hours,cultured materials were collected by centrifugation and washed withsaline two times. The cells were then suspended in 50 ml of phosphatebuffer (pH 7.6) containing 1 mM of EDTA and 0.05% of 2-mercaptethanol,and then disrupted by ultrasonic oscillation (UR-200P, TOMY SEIKO CO.).Precipitate was removed by centrifugation to obtain cell extract. To thecell extract was added protamine sulfate at a concentration of0.03%(W/V). Centrifugation was conducted as nucleic acid removaltreatment. The supernatant was subjected to salting out treatment withammonium sulfate. Fractions saturated with 20-80% ammonium sulfate werecollected and dialyzed against phosphate buffer. The dialyzed solutionwas applied to DEAE-cellulose (WHATMAN) column (diameter 50 mm×length100 mm) equilibrated with said phosphate buffer for adsorption thereof.Elution of adsorbed proteins was carried out by adding suitableconcentration of potassium chloride to said phosphate buffer.Acylglucosamine 2-epimerase activities were existed in fractions withpotassium chloride concentration of 75-100 mM, which were collected,concentrated, desalted and then applied to Q-Sepharose (PHARMACIA)(inner diameter 1 cm×length 5 cm). Elution of adsorbed proteins wascarried out by linear gradient using said phosphate buffer containing100 mM of potassium chloride and said phosphate buffer containing 300 mMof potassium chloride. Fractions with acylglucosamine 2-epimeraseactivities were collected, concentrated by said membrane filter anddesalted to obtain about 700 unit (33 mg) of purified clonedacylglucosamine 2-epimerase.

Example 4

Purification of Acylglucosamine 2-epimerase from Rat Kidney

Acylglucosamine 2-epimerase from rat kidney was purified in the samemanner as Example 1(1) using 300 g of rat kidney to give 0.15 mg ofacylglucosamine 2-epimerase, provided that acylglucosamine 2-epimerasewas purified using Mono Q column in place of reverse phase column(pBondasphere 5 μC4-300 Å) which causes inactivation of enzyme.

Example 5

Inhibition of Renin Activities by Acylglucosamine 2-epimerase (1)

A 2.5 mU of commercially available renin (SIGMA) and 0.25 pmol of clonedacylglucosamine 2-epimerase obtained in example 3 were added to 40 μl ofbuffer A (0.1M sodium phosphate buffer (pH 6.5), 1 mM EDTA, 1 μMleupeptin, 0.05% bovine serum albumin). The mixture was reacted at 37°C. for 1 hour. After the reaction, 960 μl of chilled buffer A was addedthereto to adjust the total volume thereof to 1 ml. To 250 μl of bufferA were added a part of said diluted solution thus prepared (25 μl ) and0.4 mg/ml of angiotensinogen (SIGMA) and phenylmethylsulfonyl fluorideat a final concentration of 2.5 mM. The reaction mixture was thenreacted at 37° C. for 30 minutes. The solution was treated for 3 minutesin boiled water to stop the reaction. After centrifugation (14,000 rpm,10 minutes), angiotensin I released in supernatant was determined. Theresults were shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Cloned epimerase                                                                             Remaining renin                                                  (pmol) activities (%)                                                       ______________________________________                                        0              100                                                              1 87                                                                          3 66                                                                          10 54                                                                         25 48                                                                       ______________________________________                                    

Example 6

Inhibition of Renin Activities by Acylglucosamine 2-epimerase (1)

Renin inhibition activities of acylglucosamine 2-epimerase weredetermined in the same manner as example 5 except that ratacylglucosamine 2-epimerase obtained in example 4 was employed in placeof cloned acylglucosamine 2-epimerase obtained in example 3. The resultsare shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Rat kidney epimerase                                                                          Remaining renin                                                 (pmol) activities (%)                                                       ______________________________________                                        0               100                                                             1 98                                                                          10 88                                                                         50 48                                                                       ______________________________________                                    

Example 7

Binding Reaction of Renin and Acylglucosamine 2-epimerase (1)

To 100 μl buffer A was added 25 mU of renin (SIGMA) and 140 pmol ofcloned acylglucosamine 2-epimerase obtained in example 3, and theresulting mixture was reacted at 37° C. for 1 hour.

Said reaction solution (100 μl) was fractionated by gel filtrationchromatography (Column, Superose 12HR 10/30; Mobile phase, 50 mM sodiumphosphate buffer (pH 7.5) -150 mM sodium chloride; flow rate 1 ml/min;Detection, ultraviolet (280 nm)). Renin activities of each fraction wereassayed to determine change of molecular weight of renin.

The renin activities of fractions eluted by said gel filtrationchromatography were determined according to the method of example 5.Consequently, in case of no addition of acylglucosamine 2-epimerase,renin activities were eluted at a position of about 40,000 daltonscorresponding to molecular weight of renin. However, in case of additionof acylglucosamine 2-epimerase, renin activities were eluted at aposition of about 60,000 daltons, demonstrating that said enzyme isattached to renin to increase molecular weight thereof.

The high-molecular-weight renin obtained in this example coincided veryclosely with molecular weight (about 60,000 daltons) ofhigh-molecular-weight type (HMW) renin determined by gel filtrationchromatography, i.e., the complex of renin with renin binding proteinreported by Takahashi et al (J. Biochem., 93, 1583-1594 (1983)).Therefore, said acylglucosamine 2-epimerase has renin bindingactivities.

Example 8

Binding Reaction of Renin and Acylglucosamine 2-epimerase (2)

Determination of molecular weight thereof was carried out by gelfiltration chromatography in the same manner as example 7 except thatrat acylglucosamine 2-epimerase obtained in example 4 was employed inplace of cloned acylglucosamine 2-epimerase obtained in example 3.Consequently, molecular weight of renin was increased as high as 60,000daltons as shown in example 7.

Example 9

A Method for Producing N-acetylmannosamine with Protein Having ReninBinding Activities

Production of N-acetylmannosamine was carried out from inexpensiveN-acetylglucosamine. A mixture (100 ml) consisting of 10 g ofN-acetylglucosamine, 5 mg of protein having renin binding activitiesobtained in example 3, 4 mM ATP and 10 mM MgCl₂ was adjusted at a pH of7.5, and the mixture was reacted at 37° C. for 24 hours. The reactionsolution was concentrated to give syrup in vacuo. To the syrup obtainedwas added 40 ml of ethanol and the solution was heated in a boiled waterbath for 10 minutes and then allowed to stand for 3 hours. SinceN-acetylglucosamine is insoluble and N-acetylmannosamine is insoluble,precipitate (N-acetylglucosamine) was filtered off and the filtrate wasconcentrated to give 2 g of N-acetylmannosamine with a purity of atleast 91% in vacuo.

Example 10

Production of Sialic Acid Using Protein Having Renin Binding Activities

Production of N-acetylneuraminic acid was carried out by acting theprotein having renin binding activities and N-acetylneuraminic acidlyase on N-acetylglucosamine and pyruvic acid.

A solution prepared by dissolving 22 g of N-acetylglucosamine and 11 gof pyruvic acid in 50 mM of Tris-HCl buffer (pH 7.5) containing 5 mM ATPand 5 mM MgCl₂ To the solution was added 15 mg of protein having reninbinding activities obtained in example 3 and 500 unit ofN-acetylneuraminic acid lyase, and the total volume of the solution wasadjusted to 0.5 liter. The mixture was reacted at 30° C. for 48 hours.After the reaction, 12.4 g of N-acetylneuraminic acid was produced inthe reaction mixture. Reaction product was isolated by ion-exchangechromatography with Dowex 11 (DOW CHEMICAL CO.). After concentration,10.3 g of N-acetylneuraminic acid was obtained by lyophilization.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 9                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 402 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -      (v) FRAGMENT TYPE: N-terminal                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - -  Met Glu Lys Glu Arg Glu Thr Leu Gln Ala - #Trp Lys Glu Arg Val        Gly                                                                               1               5 - #                 10 - #                 15             - -  Gln Glu Leu Asp Arg Val Met Ala Phe Trp - #Leu Glu His Ser His Asp                   20     - #             25     - #             30                  - -  Arg Glu His Gly Gly Phe Phe Thr Cys Leu - #Gly Arg Asp Gly Arg Val               35         - #         40         - #         45                      - -  Tyr Asp Asp Leu Lys Tyr Val Trp Leu Gln - #Gly Arg Gln Val Trp Met           50             - #     55             - #     60                          - -  Tyr Cys Arg Leu Tyr Arg Lys Leu Glu Arg - #Phe His Arg Pro Glu Leu       65                 - # 70                 - # 75                 - # 80       - -  Leu Asp Ala Ala Lys Ala Gly Gly Glu Phe - #Leu Leu Arg His Ala Arg                       85 - #                 90 - #                 95              - -  Val Ala Pro Pro Glu Lys Lys Cys Ala Phe - #Val Leu Thr Arg Asp Gly                   100     - #            105     - #            110                 - -  Arg Pro Val Lys Val Gln Arg Ser Ile Phe - #Ser Glu Cys Phe Tyr Thr               115         - #        120         - #        125                     - -  Met Ala Met Asn Glu Leu Trp Arg Val Thr - #Ala Glu Ala Arg Tyr Gln           130             - #    135             - #    140                         - -  Ser Glu Ala Val Asp Met Met Asp Gln Ile - #Val His Trp Val Arg Glu       145                 - #150                 - #155                 -         #160                                                                             - -  Asp Pro Ser Gly Leu Gly Arg Pro Gln Leu - #Pro Gly Ala Val Ala        Ser                                                                                              165 - #                170 - #                175            - -  Glu Ser Met Ala Val Pro Met Met Leu Leu - #Cys Leu Val Glu Gln Leu                   180     - #            185     - #            190                 - -  Gly Glu Glu Asp Glu Glu Leu Ala Gly Arg - #Tyr Ala Gln Leu Gly His               195         - #        200         - #        205                     - -  Trp Cys Ala Arg Arg Ile Leu Gln His Val - #Gln Arg Asp Gly Gln Ala           210             - #    215             - #    220                         - -  Val Leu Glu Asn Val Ser Glu Asp Gly Glu - #Glu Leu Ser Gly Cys Leu       225                 - #230                 - #235                 -         #240                                                                             - -  Gly Arg His Gln Asn Pro Gly His Ala Leu - #Glu Ala Gly Trp Phe        Leu                                                                                              245 - #                250 - #                255            - -  Leu Arg His Ser Ser Arg Ser Gly Asp Ala - #Lys Leu Arg Ala His Val                   260     - #            265     - #            270                 - -  Ile Asp Thr Phe Leu Leu Leu Pro Phe Arg - #Ser Gly Trp Asp Ala Asp               275         - #        280         - #        285                     - -  His Gly Gly Leu Phe Tyr Phe Gln Asp Ala - #Asp Gly Leu Cys Pro Thr           290             - #    295             - #    300                         - -  Gln Leu Glu Trp Ala Met Lys Leu Trp Trp - #Pro His Ser Glu Ala Met       305                 - #310                 - #315                 -         #320                                                                             - -  Ile Ala Phe Leu Met Gly Tyr Ser Glu Ser - #Gly Asp Pro Ala Leu        Leu                                                                                              325 - #                330 - #                335            - -  Arg Leu Phe Tyr Gln Val Ala Glu Tyr Thr - #Phe Arg Gln Phe Arg Asp                   340     - #            345     - #            350                 - -  Pro Glu Tyr Gly Glu Trp Phe Gly Tyr Leu - #Asn Arg Glu Gly Lys Val               355         - #        360         - #        365                     - -  Ala Leu Thr Ile Lys Gly Gly Pro Phe Lys - #Gly Cys Phe His Val Pro           370             - #    375             - #    380                         - -  Arg Cys Leu Ala Met Cys Glu Glu Met Leu - #Ser Ala Leu Leu Ser Arg       385                 - #390                 - #395                 -         #400                                                                             - -  Leu Ala                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 417 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -      (v) FRAGMENT TYPE: N-terminal                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - -  Met Glu Lys Glu Arg Glu Thr Leu Gln Ala - #Trp Lys Glu Arg Val        Gly                                                                               1               5 - #                 10 - #                 15             - -  Gln Glu Leu Asp Arg Val Val Ala Phe Trp - #Met Glu His Ser His Asp                   20     - #             25     - #             30                  - -  Gln Glu His Gly Gly Phe Phe Thr Cys Leu - #Gly Arg Glu Gly Arg Val               35         - #         40         - #         45                      - -  Tyr Asp Asp Leu Lys Tyr Val Trp Leu Gln - #Gly Arg Gln Val Trp Met           50             - #     55             - #     60                          - -  Tyr Cys Arg Leu Tyr Arg Thr Phe Glu Arg - #Phe Arg His Ala Gln Leu       65                 - # 70                 - # 75                 - # 80       - -  Leu Asp Ala Ala Lys Ala Gly Gly Glu Phe - #Leu Leu Arg Tyr Ala Arg                       85 - #                 90 - #                 95              - -  Val Ala Pro Pro Gly Lys Lys Cys Ala Phe - #Val Leu Thr Arg Asp Gly                   100     - #            105     - #            110                 - -  Arg Pro Val Lys Val Gln Arg Thr Ile Phe - #Ser Glu Cys Phe Tyr Thr               115         - #        120         - #        125                     - -  Met Ala Met Asn Glu Leu Trp Arg Ala Thr - #Gly Glu Val Arg Tyr Gln           130             - #    135             - #    140                         - -  Thr Glu Ala Val Glu Met Met Asp Gln Ile - #Val His Trp Val Gln Glu       145                 - #150                 - #155                 -         #160                                                                             - -  Asp Ala Ser Gly Leu Gly Arg Pro Gln Leu - #Gln Gly Ala Pro Ala        Ala                                                                                              165 - #                170 - #                175            - -  Glu Pro Met Ala Val Pro Met Met Leu Leu - #Asn Leu Val Glu Gln Leu                   180     - #            185     - #            190                 - -  Gly Glu Ala Asp Glu Glu Leu Ala Gly Lys - #Tyr Ala Glu Leu Gly Asp               195         - #        200         - #        205                     - -  Trp Cys Ala Arg Arg Ile Leu Gln His Val - #Gln Arg Asp Gly Gln Ala           210             - #    215             - #    220                         - -  Val Leu Glu Asn Val Ser Glu Gly Gly Lys - #Glu Leu Pro Gly Cys Leu       225                 - #230                 - #235                 -         #240                                                                             - -  Gly Arg Gln Gln Asn Pro Gly His Thr Leu - #Glu Ala Gly Trp Phe        Leu                                                                                              245 - #                250 - #                255            - -  Leu Arg His Cys Ile Arg Lys Gly Asp Pro - #Glu Leu Arg Ala His Val                   260     - #            265     - #            270                 - -  Ile Asp Lys Phe Leu Leu Leu Pro Phe His - #Ser Gly Trp Asp Pro Asp               275         - #        280         - #        285                     - -  His Gly Gly Leu Phe Tyr Phe Gln Asp Ala - #Asp Asn Phe Cys Pro Thr           290             - #    295             - #    300                         - -  Gln Leu Glu Trp Ala Met Lys Leu Trp Trp - #Pro His Ser Glu Ala Met       305                 - #310                 - #315                 -         #320                                                                             - -  Ile Ala Phe Leu Met Gly Tyr Ser Asp Ser - #Gly Asp Pro Val Leu        Leu                                                                                              325 - #                330 - #                335            - -  Arg Leu Phe Tyr Gln Val Ala Glu Tyr Thr - #Phe Arg Gln Phe Arg Asp                   340     - #            345     - #            350                 - -  Pro Glu Tyr Gly Glu Trp Phe Gly Tyr Leu - #Ser Arg Glu Gly Lys Val               355         - #        360         - #        365                     - -  Ala Leu Ser Ile Lys Gly Gly Pro Phe Lys - #Gly Cys Phe His Val Pro           370             - #    375             - #    380                         - -  Arg Cys Leu Ala Met Cys Glu Glu Met Leu - #Gly Ala Leu Leu Ser Arg       385                 - #390                 - #395                 -         #400                                                                             - -  Pro Ala Pro Ala Pro Ser Pro Ala Pro Thr - #Pro Ala Cys Arg Gly        Ala                                                                                              405 - #                410 - #                415            - -  Glu                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 419 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -      (v) FRAGMENT TYPE: N-terminal                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - -  Met Glu Lys Glu Arg Glu Thr Leu Gln Val - #Trp Lys Gln Arg Val Gly        1               5 - #                 10 - #                 15              - -  Gln Glu Leu Asp Ser Val Ile Ala Phe Trp - #Met Glu His Ser His Asp                   20     - #             25     - #             30                  - -  Gln Glu His Gly Gly Phe Phe Thr Cys Leu - #Gly Arg Asp Gly Gln Val               35         - #         40         - #         45                      - -  Tyr Asp His Leu Lys Tyr Val Trp Leu Gln - #Gly Arg Gln Val Trp Met           50             - #     55             - #     60                          - -  Tyr Cys Arg Leu Tyr Arg Thr Phe Glu Arg - #Phe Arg Arg Val Glu Leu       65                 - # 70                 - # 75                 - # 80       - -  Leu Asp Ala Ala Lys Ala Gly Gly Glu Phe - #Leu Leu Ser Tyr Ala Arg                       85 - #                 90 - #                 95              - -  Val Ala Pro Pro Gly Lys Lys Cys Ala Phe - #Val Leu Thr Gln Asp Gly                   100     - #            105     - #            110                 - -  Arg Pro Val Lys Val Gln Arg Thr Ile Phe - #Ser Glu Cys Phe Tyr Thr               115         - #        120         - #        125                     - -  Met Ala Met Asn Glu Leu Trp Lys Val Thr - #Gly Glu Met His Tyr Gln           130             - #    135             - #    140                         - -  Arg Glu Ala Val Glu Met Met Asp Gln Ile - #Ile His Trp Val Arg Glu       145                 - #150                 - #155                 -         #160                                                                             - -  Asp Pro Ala Gly Leu Gly Arg Pro Gln Leu - #Ser Gly Thr Leu Ala        Thr                                                                                              165 - #                170 - #                175            - -  Glu Pro Met Ala Val Pro Met Met Leu Leu - #Asn Leu Val Glu Gln Leu                   180     - #            185     - #            190                 - -  Gly Glu Glu Asp Glu Glu Met Thr Asp Lys - #Tyr Ala Glu Leu Gly Asp               195         - #        200         - #        205                     - -  Trp Cys Ala His Arg Ile Leu Gln His Val - #Gln Arg Asp Gly Gln Val           210             - #    215             - #    220                         - -  Val Leu Glu Asn Val Ser Glu Asp Gly Lys - #Glu Leu Pro Gly Cys Leu       225                 - #230                 - #235                 -         #240                                                                             - -  Gly Arg His Gln Asn Pro Gly His Thr Leu - #Glu Ala Gly Trp Phe        Leu                                                                                              245 - #                250 - #                255            - -  Leu Gln Tyr Ala Leu Arg Lys Gly Asp Pro - #Lys Leu Gln Arg His Ile                   260     - #            265     - #            270                 - -  Ile Asp Lys Phe Leu Leu Leu Pro Phe His - #Ser Gly Trp Asp Pro Glu               275         - #        280         - #        285                     - -  His Gly Gly Leu Phe Tyr Phe Gln Asp Ala - #Asp Asp Leu Cys Pro Thr           290             - #    295             - #    300                         - -  Gln Leu Glu Trp Asn Met Lys Leu Trp Trp - #Pro His Thr Glu Ala Met       305                 - #310                 - #315                 -         #320                                                                             - -  Ile Ala Phe Leu Met Gly Tyr Arg Asp Ser - #Gly Asp Pro Ala Leu        Leu                                                                                              325 - #                330 - #                335            - -  Asn Leu Phe Tyr Gln Val Ala Glu Tyr Thr - #Phe His Gln Phe Arg Asp                   340     - #            345     - #            350                 - -  Pro Glu Tyr Gly Glu Trp Phe Gly Tyr Leu - #Asn Gln Glu Gly Lys Val               355         - #        360         - #        365                     - -  Ala Leu Thr Ile Lys Gly Gly Pro Phe Lys - #Gly Cys Phe His Val Pro           370             - #    375             - #    380                         - -  Arg Cys Leu Ala Met Cys Glu Gln Ile Leu - #Gly Ala Leu Leu Gln Arg       385                 - #390                 - #395                 -         #400                                                                             - -  Leu Gly Pro Ala Pro Leu Gly Ser Leu Pro - #Ala Val Pro Thr Arg        Glu                                                                                              405 - #                410 - #                415            - -  Gly Ser Lys                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 402 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -      (v) FRAGMENT TYPE: N-terminal                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - -  Met Glu Lys Glu Arg Glu Thr Leu Gln Ala - #Trp Lys Glu Arg Val Gly        1               5 - #                 10 - #                 15              - -  Gln Glu Leu Asp Arg Val Met Ala Phe Trp - #Leu Glu His Ser His Asp                   20     - #             25     - #             30                  - -  Arg Glu His Gly Gly Phe Phe Thr Cys Leu - #Gly Arg Asp Gly Arg Val               35         - #         40         - #         45                      - -  Tyr Asp Asp Leu Lys Tyr Val Trp Leu Gln - #Gly Arg Gln Val Trp Met           50             - #     55             - #     60                          - -  Tyr Cys Arg Leu Tyr Arg Lys Leu Glu Arg - #Phe His Arg Pro Glu Leu       65                 - # 70                 - # 75                 - # 80       - -  Leu Asp Ala Ala Lys Ala Gly Gly Glu Phe - #Leu Leu Arg His Ala Arg                       85 - #                 90 - #                 95              - -  Val Ala Pro Pro Glu Lys Lys Cys Ala Phe - #Val Leu Thr Arg Asp Gly                   100     - #            105     - #            110                 - -  Arg Pro Val Lys Val Gln Arg Ser Ile Phe - #Ser Glu Cys Phe Tyr Thr               115         - #        120         - #        125                     - -  Met Ala Met Asn Glu Leu Trp Arg Val Thr - #Ala Glu Ala Arg Tyr Gln           130             - #    135             - #    140                         - -  Ser Glu Ala Val Glu Met Met Asp Gln Ile - #Val His Trp Val Arg Glu       145                 - #150                 - #155                 -         #160                                                                             - -  Asp Pro Ser Gly Leu Gly Arg Pro Gln Leu - #Pro Gly Ala Val Ala        Ser                                                                                              165 - #                170 - #                175            - -  Glu Ser Met Ala Val Pro Met Met Leu Leu - #Cys Leu Val Glu Gln Leu                   180     - #            185     - #            190                 - -  Gly Glu Glu Asp Glu Glu Leu Ala Gly Arg - #Tyr Ala Gln Leu Gly His               195         - #        200         - #        205                     - -  Trp Cys Ala Arg Arg Ile Leu Gln His Val - #Gln Arg Asp Gly Gln Ala           210             - #    215             - #    220                         - -  Val Leu Glu Asn Val Ser Glu Asp Gly Glu - #Glu Leu Ser Gly Cys Leu       225                 - #230                 - #235                 -         #240                                                                             - -  Gly Arg His Gln Asn Pro Gly His Ala Leu - #Glu Ala Gly Trp Phe        Leu                                                                                              245 - #                250 - #                255            - -  Leu Arg His Ser Ser Arg Ser Gly Asp Ala - #Lys Leu Arg Ala His Val                   260     - #            265     - #            270                 - -  Ile Asp Thr Phe Leu Leu Leu Pro Phe Arg - #Ser Gly Trp Asp Ala Asp               275         - #        280         - #        285                     - -  Tyr Gly Gly Leu Phe Tyr Phe Gln Asp Ala - #Asp Gly Leu Cys Pro Thr           290             - #    295             - #    300                         - -  Gln Leu Glu Trp Ala Met Lys Leu Trp Trp - #Pro His Arg Gln Ala Met       305                 - #310                 - #315                 -         #320                                                                             - -  Ile Ala Phe Leu Met Gly Tyr Ser Glu Ser - #Gly Asp Pro Ala Leu        Leu                                                                                              325 - #                330 - #                335            - -  Arg Leu Phe Tyr Gln Val Ala Glu Tyr Thr - #Phe Arg Gln Phe Arg Asp                   340     - #            345     - #            350                 - -  Pro Glu Tyr Gly Glu Trp Phe Gly Tyr Leu - #Asn Arg Glu Gly Lys Val               355         - #        360         - #        365                     - -  Ala Leu Thr Ile Lys Gly Gly Pro Phe Lys - #Gly Cys Phe His Val Pro           370             - #    375             - #    380                         - -  Arg Cys Leu Ala Met Cys Glu Glu Met Leu - #Ser Ala Leu Leu Ser Arg       385                 - #390                 - #395                 -         #400                                                                             - -  Leu Ala                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1209 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - -  ATGGAGAAGG AGCGCGAAAC TCTGCAGGCC TGGAAGGAGC GTGTGGGCCA - #AGAGCTGG    AC    60                                                                        - -  CGCGTGATGG CTTTCTGGCT GGAGCACTCC CACGATCGGG AGCACGGGGG - #CTTCTTCAC    G   120                                                                         - -  TGCCTGGGCC GCGACGGGCG GGTGTATGAC GACCTCAAGT ACGTCTGGCT - #GCAGGGGAG    G   180                                                                         - -  CAGGTGTGGA TGTACTGTCG CCTGTACCGC AAGCTTGAGC GCTTCCACCG - #CCCTGAGCT    T   240                                                                         - -  CTGGATGCGG CTAAAGCAGG GGGCGAATTT TTGCTGCGCC ATGCCCGAGT - #GGCACCTCC    T   300                                                                         - -  GAAAAGAAGT GTGCCTTTGT GCTGACGCGG GACGGCCGGC CCGTCAAGGT - #GCAGCGGAG    C   360                                                                         - -  ATCTTCAGTG AGTGCTTCTA CACCATGGCC ATGAACGAGC TGTGGAGGGT - #GACGGCGGA    G   420                                                                         - -  GCACGGTACC AGAGCGAAGC GGTGGACATG ATGGATCAGA TCGTGCACTG - #GGTGCGAGA    G   480                                                                         - -  GACCCCTCTG GGCTGGGCCG GCCCCAGCTC CCCGGGGCCG TGGCCTCGGA - #GTCCATGGC    A   540                                                                         - -  GTGCCCATGA TGCTGCTGTG CCTGGTGGAG CAGCTCGGGG AGGAGGACGA - #GGAGCTGGC    A   600                                                                         - -  GGCCGCTACG CGCAGCTGGG GCACTGGTGC GCTCGGAGGA TCCTGCAGCA - #CGTCCAGAG    G   660                                                                         - -  GATGGACAGG CTGTGCTGGA GAATGTGTCG GAAGATGGCG AGGAACTTTC - #TGGCTGCCT    G   720                                                                         - -  GGGAGACACC AGAACCCAGG CCACGCGCTG GAAGCTGGCT GGTTCCTGCT - #CCGCCACAG    C   780                                                                         - -  AGCCGGAGCG GTGACGCCAA ACTTCGAGCC CACGTCATCG ACACGTTCCT - #GCTACTGCC    T   840                                                                         - -  TTCCGCTCCG GATGGGACGC TGATCACGGA GGCCTCTTCT ACTTCCAGGA - #TGCCGATGG    C   900                                                                         - -  CTCTGCCCCA CCCAGCTGGA GTGGGCCATG AAGCTCTGGT GGCCGCACAG - #CGAAGCCAT    G   960                                                                         - -  ATCGCCTTTC TCATGGGCTA CAGTGAGAGC GGGGACCCTG CCTTACTGCG - #TCTCTTCTA    C  1020                                                                         - -  CAGGTGGCCG AGTACACGTT TCGCCAGTTT CGTGATCCCG AGTACGGGGA - #ATGGTTTGG    C  1080                                                                         - -  TACCTGAACC GAGAGGGGAA GGTTGCCCTC ACTATCAAGG GGGGTCCCTT - #TAAAGGCTG    C  1140                                                                         - -  TTCCACGTGC CGCGGTGCCT TGCCATGTGC GAAGAGATGC TGAGCGCCCT - #GCTGAGCCG    C  1200                                                                         - -  CTCGCCTAG               - #                  - #                       - #       1209                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -      (v) FRAGMENT TYPE: internal                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - -  Pro Ala Pro Ser Pro Ala Pro Thr Pro Ala - #Cys Arg Gly Ala Glu           1               5 - #                 10 - #                 15              - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -      (v) FRAGMENT TYPE: internal                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - -  Pro Ala Pro Leu Gly Ser Leu Pro Ala Val - #Pro Thr Arg Glu Gly Ser        1               5 - #                 10 - #                 15              - -  Lys                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -      (v) FRAGMENT TYPE: internal                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - -  Lys Gly Asn Lys Ser Trp Gln Asp                                           1               5                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1209 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Coding Se - #quence                                             (B) LOCATION: 1...1206                                                        (D) OTHER INFORMATION:                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - -  ATG GAG AAG GAG CGC GAA ACT CTG CAG GCC - #TGG AAG GAG CGT GTG GGC          48                                                                         Met Glu Lys Glu Arg Glu Thr Leu Gln Ala - #Trp Lys Glu Arg Val Gly             1               5 - #                 10 - #                 15              - -  CAA GAG CTG GAC CGC GTG ATG GCT TTC TGG - #CTG GAG CAC TCC CAC GAT          96                                                                         Gln Glu Leu Asp Arg Val Met Ala Phe Trp - #Leu Glu His Ser His Asp                        20     - #             25     - #             30                  - -  CGG GAG CAC GGG GGC TTC TTC ACG TGC CTG - #GGC CGC GAC GGG CGG GTG         144                                                                         Arg Glu His Gly Gly Phe Phe Thr Cys Leu - #Gly Arg Asp Gly Arg Val                    35         - #         40         - #         45                      - -  TAT GAC GAC CTC AAG TAC GTC TGG CTG CAG - #GGG AGG CAG GTG TGG ATG         192                                                                         Tyr Asp Asp Leu Lys Tyr Val Trp Leu Gln - #Gly Arg Gln Val Trp Met                50             - #     55             - #     60                          - -  TAC TGT CGC CTG TAC CGC AAG CTT GAG CGC - #TTC CAC CGC CCT GAG CTT         240                                                                         Tyr Cys Arg Leu Tyr Arg Lys Leu Glu Arg - #Phe His Arg Pro Glu Leu            65                 - # 70                 - # 75                 - # 80       - -  CTG GAT GCG GCT AAA GCA GGG GGC GAA TTT - #TTG CTG CGC CAT GCC CGA         288                                                                         Leu Asp Ala Ala Lys Ala Gly Gly Glu Phe - #Leu Leu Arg His Ala Arg                            85 - #                 90 - #                 95              - -  GTG GCA CCT CCT GAA AAG AAG TGT GCC TTT - #GTG CTG ACG CGG GAC GGC         336                                                                         Val Ala Pro Pro Glu Lys Lys Cys Ala Phe - #Val Leu Thr Arg Asp Gly                        100     - #            105     - #            110                 - -  CGG CCC GTC AAG GTG CAG CGG AGC ATC TTC - #AGT GAG TGC TTC TAC ACC         384                                                                         Arg Pro Val Lys Val Gln Arg Ser Ile Phe - #Ser Glu Cys Phe Tyr Thr                    115         - #        120         - #        125                     - -  ATG GCC ATG AAC GAG CTG TGG AGG GTG ACG - #GCG GAG GCA CGG TAC CAG         432                                                                         Met Ala Met Asn Glu Leu Trp Arg Val Thr - #Ala Glu Ala Arg Tyr Gln                130             - #    135             - #    140                         - -  AGC GAA GCG GTG GAC ATG ATG GAT CAG ATC - #GTG CAC TGG GTG CGA GAG         480                                                                         Ser Glu Ala Val Asp Met Met Asp Gln Ile - #Val His Trp Val Arg Glu            145                 - #150                 - #155                 -         #160                                                                             - -  GAC CCC TCT GGG CTG GGC CGG CCC CAG CTC - #CCC GGG GCC GTG GCC        TCG     528                                                                      Asp Pro Ser Gly Leu Gly Arg Pro Gln Leu - #Pro Gly Ala Val Ala Ser                           165 - #                170 - #                175             - -  GAG TCC ATG GCA GTG CCC ATG ATG CTG CTG - #TGC CTG GTG GAG CAG CTC         576                                                                         Glu Ser Met Ala Val Pro Met Met Leu Leu - #Cys Leu Val Glu Gln Leu                        180     - #            185     - #            190                 - -  GGG GAG GAG GAC GAG GAG CTG GCA GGC CGC - #TAC GCG CAG CTG GGG CAC         624                                                                         Gly Glu Glu Asp Glu Glu Leu Ala Gly Arg - #Tyr Ala Gln Leu Gly His                    195         - #        200         - #        205                     - -  TGG TGC GCT CGG AGG ATC CTG CAG CAC GTC - #CAG AGG GAT GGA CAG GCT         672                                                                         Trp Cys Ala Arg Arg Ile Leu Gln His Val - #Gln Arg Asp Gly Gln Ala                210             - #    215             - #    220                         - -  GTG CTG GAG AAT GTG TCG GAA GAT GGC GAG - #GAA CTT TCT GGC TGC CTG         720                                                                         Val Leu Glu Asn Val Ser Glu Asp Gly Glu - #Glu Leu Ser Gly Cys Leu            225                 - #230                 - #235                 -         #240                                                                             - -  GGG AGA CAC CAG AAC CCA GGC CAC GCG CTG - #GAA GCT GGC TGG TTC        CTG     768                                                                      Gly Arg His Gln Asn Pro Gly His Ala Leu - #Glu Ala Gly Trp Phe Leu                           245 - #                250 - #                255             - -  CTC CGC CAC AGC AGC CGG AGC GGT GAC GCC - #AAA CTT CGA GCC CAC GTC         816                                                                         Leu Arg His Ser Ser Arg Ser Gly Asp Ala - #Lys Leu Arg Ala His Val                        260     - #            265     - #            270                 - -  ATC GAC ACG TTC CTG CTA CTG CCT TTC CGC - #TCC GGA TGG GAC GCT GAT         864                                                                         Ile Asp Thr Phe Leu Leu Leu Pro Phe Arg - #Ser Gly Trp Asp Ala Asp                    275         - #        280         - #        285                     - -  CAC GGA GGC CTC TTC TAC TTC CAG GAT GCC - #GAT GGC CTC TGC CCC ACC         912                                                                         His Gly Gly Leu Phe Tyr Phe Gln Asp Ala - #Asp Gly Leu Cys Pro Thr                290             - #    295             - #    300                         - -  CAG CTG GAG TGG GCC ATG AAG CTC TGG TGG - #CCG CAC AGC GAA GCC ATG         960                                                                         Gln Leu Glu Trp Ala Met Lys Leu Trp Trp - #Pro His Ser Glu Ala Met            305                 - #310                 - #315                 -         #320                                                                             - -  ATC GCC TTT CTC ATG GGC TAC AGT GAG AGC - #GGG GAC CCT GCC TTA        CTG    1008                                                                      Ile Ala Phe Leu Met Gly Tyr Ser Glu Ser - #Gly Asp Pro Ala Leu Leu                           325 - #                330 - #                335             - -  CGT CTC TTC TAC CAG GTG GCC GAG TAC ACG - #TTT CGC CAG TTT CGT GAT        1056                                                                         Arg Leu Phe Tyr Gln Val Ala Glu Tyr Thr - #Phe Arg Gln Phe Arg Asp                        340     - #            345     - #            350                 - -  CCC GAG TAC GGG GAA TGG TTT GGC TAC CTG - #AAC CGA GAG GGG AAG GTT        1104                                                                         Pro Glu Tyr Gly Glu Trp Phe Gly Tyr Leu - #Asn Arg Glu Gly Lys Val                    355         - #        360         - #        365                     - -  GCC CTC ACT ATC AAG GGG GGT CCC TTT AAA - #GGC TGC TTC CAC GTG CCG        1152                                                                         Ala Leu Thr Ile Lys Gly Gly Pro Phe Lys - #Gly Cys Phe His Val Pro                370             - #    375             - #    380                         - -  CGG TGC CTT GCC ATG TGC GAA GAG ATG CTG - #AGC GCC CTG CTG AGC CGC        1200                                                                         Arg Cys Leu Ala Met Cys Glu Glu Met Leu - #Ser Ala Leu Leu Ser Arg            385                 - #390                 - #395                 -         #400                                                                             - -  CTC GCC TAG             - #                  - #                      - #       1209                                                                  Leu Ala                                                                     __________________________________________________________________________

What is claimed is:
 1. A purified acylglucosamine 2-epimerase selectedfrom the group consisting of:(1) a polypeptide having the amino acidsequence listed as SEQ ID NO:1; (2) a polypeptide having an amino acidsequence of SEQ ID NO:1 from which at least one position selected fromthe group consisting of 10, 13, 21, 23, 27, 33, 45, 47, 51, 71, 72,76-79, 93, 94, 101, 110, 120, 136, 137, 139, 141, 142, 145, 149, 155,159, 162, 163, 171, 173, 174, 176, 178, 187, 195, 199-202, 205, 208,212, 224, 232, 234, 237, 243, 249, 258-261, 263, 266, 267, 269, 270,272,275, 282, 287-289, 300, 301, 309, 317, 318, 328, 329, 334, 337, 348,363, 364, 371, 392, 393, 395, 399, 401, and 402 is eliminated orreplaced with another amino acid; and (3) a polypeptide having: (a) apartial amino acid sequence of SEQ ID NO:1 wherein a polypeptide havingsaid partial amino acid sequence has acylglucosamine 2-epimeraseactivity; and (b) an amino acid sequence selected from the groupconsisting of SEQ ID NO:6, NO:7, and NO:8, wherein a peptide havingsequence (b) is attached to the N-terminal or C-terminal of thepolypeptide having sequence (a).
 2. An antihypertensive agentcomprising, as an essential component, a purified acylglucosamine2-epimerase according to claim 1, and pharmaceutically acceptablecarrier.
 3. An isolated polypeptide having an amino acid sequence otherthan the amino acid sequence listed as SEQ ID NO:2, NO:3, or NO:4,comprising an amino acid sequence selected from the group consistingof:(1) the amino acid sequence listed as SEQ ID NO:1; (2) an amino acidsequence of SEQ ID NO:1 from which at least one position selected fromthe group consisting of 10, 13, 21, 23, 27, 33, 45, 47, 51, 71, 72,76-79, 93, 94, 101, 110, 120, 136, 137, 139, 141, 142, 145, 149, 155,159 162, 163, 171, 173, 174, 176, 178, 187, 195, 199-202, 205, 208, 212,224, 232, 234, 237, 243, 249, 258-261, 263, 266, 267, 269, 270,272, 275,282, 287-289,300, 301, 309, 317, 318, 328, 329, 334, 337, 348, 363, 364,371, 392, 393, 395, 399, 401, and 402 is eliminated or replaced withanother amino acid; and (3) a combination of: (a) a partial amino acidsequence of SEQ ID NO:1 wherein a polypeptide having said partial aminoacid sequence has acylglucosamine 2-epimerase activity; and (b) an aminoacid sequence selected from the group consisting of SEQ ID NO:6, NO:7,and NO:8, wherein a peptide having sequence (b) is attached to theN-terminal or C-terminal of the polypeptide having sequence (a).
 4. Anepimerizing agent converting N-acetylglucosamine to N-acetylmannosaminecomprising, as an essential component, a purified polypeptide selectedfrom the group consisting of polypeptides according to claim 3 andpolypeptides having the amino acid sequence listed as SEQ ID NO:2, NO:3,or NO:4.
 5. A purified epimerase having renin-binding activitiescomprising a polypeptide selected from the group consisting of:(1) apolypeptide having the amino acid sequence listed as SEQ ID NO:1; (2) apolypeptide having an amino acid sequence of SEQ ID NO:1 from which atleast one position selected from the group consisting of 10, 13, 21, 23,27, 33, 45, 47, 51, 71, 72, 76-79, 93, 94, 101, 110, 120, 136, 137, 139,141, 142, 145, 149, 155, 159, 162, 163, 171, 173, 174, 176, 178, 187,195, 199-202, 205, 208, 212, 224, 232, 234, 237, 243, 249, 258-261, 263,266, 267, 269, 270, 272, 275, 282, 287-289, 300, 301, 309, 317, 318,328, 329, 334, 337, 348, 363, 364, 371, 392, 393, 395, 399, 401, and 402is eliminated or replaced with another amino acid; and (3) a polypeptidehaving: (a) a partial amino acid sequence of SEQ ID NO:1 wherein apolypeptide having said partial amino acid sequence has acylglucosamine2-epimerase activity; and (b) an amino acid sequence selected from thegroup consisting of SEQ ID NO:6, NO:7, and NO:8, wherein a peptidehaving sequence (b) is attached to the N-terminal or C-terminal of thepolypeptide having sequence (a).
 6. A method for producingN-acetylmannosamine comprising causing a polypeptide havingrenin-binding activity to act on N-acetylglucosamine, wherein saidpolypeptide is selected from the group consisting of:(A) isolatedpolypeptides having an amino acid sequence other than the amino acidsequence listed as SEQ ID NO:2, NO:3, or NO:4, comprising an amino acidsequence selected from the group consisting of:(i) the amino acidsequence listed as SEQ ID NO:1; (ii) an amino acid sequence of SEQ IDNO:1 from which at least one position selected from the group consistingof 10, 13, 21, 23, 27, 33, 45, 47, 51, 71, 72, 76-79, 93, 94, 101, 110,120, 136, 137, 139, 141, 142, 145, 149, 155, 159, 162, 163, 171, 173,174, 176, 178, 187, 195, 199-202, 205, 208, 212, 224, 232, 234, 237,243, 249, 258-261, 263, 266, 267, 269, 270, 272, 275, 282, 287-289, 300,301, 309, 317, 318, 328, 329, 334, 337, 348, 363, 364, 371, 392, 393,395, 399, 401, and 402 is eliminated or replaced with another aminoacid; and (iii) a combination of: (a) a partial amino acid sequence ofSEQ ID NO:1 wherein a polypeptide having said partial amino acidsequence has acylglucosamine 2-epimerase activity; and (b) an amino acidsequence selected from the group consisting of SEQ ID NO:6, NO:7, andNO:8, wherein a peptide having sequence (b) is attached to theN-terminal or C-terminal of the polypeptide having sequence (a), and (B)isolated polypeptides having the amino acid sequence listed as SEQ IDNO:2, NO:3, or NO:4.
 7. A method for producing N-acetylneuraminic acidcomprising causing a polypeptide having renin-binding activity andN-acetylneuraminic acid lyase to act on N-acetylglucosamine and pyruvicacid, wherein said polypeptide is selected from the group consistingof:(A) isolated polypeptides having an amino acid sequence other thanthe amino acid sequence listed as SEQ ID NO:2, NO:3, or NO:4, comprisingan amino acid sequence selected from the group consisting of:(i) theamino acid sequence listed as SEQ ID NO:1; (ii) an amino acid sequenceof SEQ ID NO:1 from which at least one position selected from the groupconsisting of 10, 13, 21, 23, 27, 33, 45, 47, 51, 71, 72, 76-79, 93, 94,101, 110, 120, 136, 137, 139, 141, 142, 145, 149, 155, 159, 162, 163,171, 173, 174, 176, 178, 187, 195, 199-202, 205, 208, 212, 224, 232,234, 237, 243, 249, 258-261, 263, 266, 267, 269, 270, 272, 275, 282,287-289, 300, 301, 309, 317, 318, 328, 329, 334, 337, 348, 363, 364,371, 392, 393, 395, 399, 401, and 402 is eliminated or replaced withanother amino acid; and (iii) a combination of: (a) a partial amino acidsequence of SEQ ID NO:1 wherein a polypeptide having said partial aminoacid sequence has acylglucosamine 2-epimerase activity; and (b) an aminoacid sequence selected from the group consisting of SEQ ID NO:6, NO:7,and NO:8, wherein a peptide having sequence (b) is attached to theN-terminal or C-terminal of the polypeptide having sequence (a), and (B)isolated polypeptides having the amino acid sequence listed as SEQ IDNO:2, NO:3, or NO:4.